Hepatitis C virus non-structural NS3/4A fusion gene

ABSTRACT

Disclosed herein is the discovery of a novel hepatitis C virus (HCV) isolated from a human patient. Embodiments of the invention include HCV peptides, nucleic acids encoding said HCV peptides, antibodies directed to said peptides, compositions containing said nucleic acids and peptides, as well as, methods of making and using the aforementioned compositions including, but not limited to, diagnostics and medicaments for the treatment and prevention of HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 09/930,591, filed Aug. 15, 2001, now U.S. Pat. No.6,960,569 which claims the benefit of priority to U.S. ProvisionalPatent Application No. 60/225,767, filed Aug. 17, 2000, and U.S.Provisional Patent Application No. 60/229,175, filed Aug. 29, 2000; theentire disclosures of the afore-mentioned patent application andprovisional applications are hereby expressly incorporated by referencein their entireties.

FIELD OF THE INVENTION

The present invention relates to the discovery of a novel hepatitis Cvirus (HCV) isolated from a human patient. Embodiments include novel HCVpeptides, nucleic acids encoding said HCV peptides, antibodies directedto said peptides, compositions containing said nucleic acids andpeptides, as well as methods of making and using the aforementionedcompositions including, but not limited to, diagnostics and medicamentsfor the treatment and prevention of HCV infection.

BACKGROUND OF THE INVENTION

Viruses are intracellular parasites that require the biochemicalmachinery of a host cell for replication and propagation. All virusparticles contain some genetic information that encodes viral structuralproteins and enzymes. The genetic material may be DNA or RNA, in double-or single stranded form. (Virology, Fields ed., third edition,Lippencott-Raven publishers, pp 72-83 (1996)). The viral nucleic acid issurrounded by a coat of proteins called the capsid. (Id.) In someviruses the capsid is surrounded by an additional layer comprised of alipid membrane, referred to as the envelope. (Id. at 83-95).

The typical viral life cycle begins with infection of a host cellthrough attachment of the virus particle to a cell surface receptor andinternalization of the viral capsid. (Id. at 103). Accordingly, a virus'host range is limited to cells that express an appropriate cell surfacereceptor. Once internalized, the virus particle is disassembled and itsnucleic acid is transcribed, translated or replicated. (Id.) At thispoint, the virus may undergo lytic replication, where new virusparticles are formed and released from the infected cell. (Id. at105-11). The Influenza virus is a typical example of a virus thatundergoes lytic replication immediately upon infection of a host cell.(Id. at 1369-85).

Alternatively, a virus may enter a latent phase, referred to aslysogeny, where the genome is replicated but few if any viral proteinsare actually expressed and viral particles are not formed. (Id. at219-29). Herpesviruses such as the Epstein-Barr Virus are typicalexamples of viruses that establish latent infection in the host cells.(Id. at 229-34). Eventually, in order for the virus to spread, it mustexit lysogeny and enter the lytic phase. The viral particles that arereleased during the lytic phase infect other cells of the sameindividual or can be transmitted to another individual where a newinfection is established.

Since the viral life cycle comprises both an intracellular andextracellular phase, both the humoral and cell-mediated immune defensesystems are important for combating viral infections. (Id. at 467-73).Antibodies directed against viral proteins may block the virusparticle's interaction with its cellular receptor or otherwise interferewith the internalization or release processes. (Id. at 471). An antibodycapable of interfering with the viral life cycle is referred to as aneutralizing antibody.

During intracellular replication, viral proteins, which are foreign tothe host cell, are produced and some of these proteins are digested bycellular proteases after coupling to a Major Histocompatibility Complex(MHC) molecule presented on the surface of the infected cell. (Id. at350-58). Thus, the infected cell is recognized by T-lymphocytes,macrophages or NK-cells and killed before the virus replicates andspreads to adjacent cells. (Id. at 468-70). In addition, the presence ofviral nucleic acids, most notably as double-stranded RNA, triggers theinfected cell to shut down its translation machinery and to produceantiviral signaling molecules known as interferons. (Id. at 376-79).

Viruses have evolved various means of evading the immune defense systemof the host, however. By establishing latency (i.e., lysogeny), forexample, the virus does not enter the lytic phase and avoids the humoralimmune defense system. (Id. at 224). During the latent phase, few viralproteins are produced and infected cells have only a minimal ability topresent evidence to surrounding lymphocytes and macrophages of theirinfected state. (Id. at 225-26). Additionally, some viral proteins, mostnotably those produced during latency, evolve polypeptide sequences thatcannot be efficiently presented to the cell mediated immune defensesystem. (Levitskaya et al., Nature 375:685-88 (1995)). Finally, someviruses may actively interfere with the immune response of the infectedhost, for instance by preventing surface expression of MHC molecules(Fruh et al., J. Mol. Med. 75:18-27 (1997)), or by disrupting interferonsignaling (Fortunato et al., Trends Microbiol. 8:111-19 (2000)).

Particularly evasive are the hepatitis viruses, which are not classifiedas a family but are grouped based on their ability to infect cells ofthe liver. Hepatitis C Virus (HCV) belongs to the Flaviviridae family ofsingle-stranded RNA viruses. (Virology, Fields ed., third edition,Lippencott-Raven publishers, pp 945-51 (1996)). The HCV genome isapproximately 9.6 kb in length, and encodes at least ten polypeptides.(Kato, Microb. Comp. Genomics, 5:129-151 (2000)). The genomic RNA istranslated into one single polyprotein that is subsequently cleaved byviral and cellular proteases to yield the functional polypeptides. (Id.)The polyprotein is cleaved to three structural proteins (core protein,E1 and E2), to p7 of unknown function, and to six non-structural (NS)proteins (NS2, NS3, NS4A/B, NS5A/B). (Id.) NS3 encodes a serine proteasethat is responsible for some of the proteolytic events required forvirus maturation (Kwong et al., Antiviral Res., 41:67-84 (1999)) andNS4A acts as a co-factor for the NS3 protease. (Id.) NS3 furtherdisplays NTPase activity, and possesses RNA helicase activity in vitro.(Kwong et al., Curr. Top. Microbiol. Immunol., 242:171-96 (2000)).

HCV infection typically progresses from an acute to a chronic phase.(Virology, Fields ed., third edition, Lippencott-Raven publishers, pp1041-47 (1996)). Acute infection is characterized by high viralreplication and high viral load in liver tissue and peripheral blood.(Id. at 1041-42.) The acute infection is cleared by the patient's immunedefense system in roughly 15% of the infected individuals; in the other85% the virus establishes a chronic, persistent infection. (Lawrence,Adv. Intern. Med., 45:65-105 (2000)). During the chronic phasereplication takes place in the liver, and some virus can be detected inperipheral blood. (Virology, Fields ed., third edition, Lippencott-Ravenpublishers, pp 1042 (1996)).

Essential to the establishment of a persistent infection is theevolution of strategies for evading the host's immune defense system.HCV, as a single stranded RNA virus, displays a high mutation rate inthe replication and transcription of its genome. (Id. at 1046). Thus, ithas been noted that the antibodies produced during the lytic phaseseldom neutralize virus strains produced during chronic infection. (Id.)Although it appears HCV is not interfering with antigen processing andpresentation on MHC-I molecules, the viral NS5A protein may be involvedin repression of interferon signaling through inhibition of the PKRprotein kinase. (Tan et al., Virology, 284:1-12 (2001)).

The infected host mounts both a humoral and a cellular immune responseagainst the HCV virus but in most cases the response fails to preventestablishment of the chronic disease. Following the acute phase, theinfected patient produces antiviral antibodies including neutralizingantibodies to the envelope proteins E1 and E2. (Id. at 1045). Thisantibody response is sustained during chronic infection. (Id.) Inchronically infected patients, the liver is also infiltrated by bothCD8+ and CD4+ lymphocytes. (Id. at 1044-45). Additionally, infectedpatients produce interferons as an early response to the viralinfection. (Id. at 1045). It is likely that the vigor of the initialimmune response against the infection determines whether the virus willbe cleared or whether the infection will progress to a chronic phase.(Pape et al., J. Viral. Hepat., 6 Supp. 1:36-40 (1999)). Despite theefforts of others, the need for efficient immunogens and medicaments forthe prevention and treatment of HCV infection is manifest.

SUMMARY OF THE INVENTION

A new HCV sequence was discovered. A novel NS3/4A fragment of the HCVgenome was cloned and sequenced from a patient infected with HCV (SEQ.ID. NO.: 1). This sequence was found to be only 93% homologous to themost closely related HCV sequence. Embodiments include this novelpeptide (SEQ. ID. NO.: 2) and fragments thereof at least 3, 4, 6, 8, 10,12, 15 or 20 amino acids in length, nucleic acids encoding thesemolecules, vectors having said nucleic acids, and cells having saidvectors, nucleic acids, or peptides. The NS3/4A nucleic acid, fragmentsthereof and corresponding peptides are immunogenic. Accordingly,preferred embodiments include vaccine compositions comprising the HCVpeptide of SEQ. ID. NO.: 2 or a fragment thereof at least 3, 4, 6, 8,10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 14 and 15)or a nucleic acid encoding said peptide or fragments.

Mutants of the NS3/4A peptide were also created. Some mutants aretruncated versions of the NS3/4A peptide (e.g., SEQ. ID. NOs.: 12 and13) and others lack a proteolytic cleavage site (e.g., SEQ. ID. NOs.:3-11). These molecules and the nucleic acids encoding them are alsoimmunogenic. These novel peptides (SEQ. ID. NOs.: 3-13) and fragmentsthereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length(e.g., SEQ. ID. NOs.: 15-26), nucleic acids encoding these molecules,vectors having said nucleic acids, and cells having said vectors,nucleic acids, or peptides are embodiments of the invention. Aparticularly preferred embodiment is a vaccine composition comprising atleast one HCV peptide of SEQ. ID. NOs.: 3-11 or a fragment thereof atleast 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID.NOs.: 16-26) or a nucleic acid encoding said peptides or fragments.

Methods of making and using the compositions described herein are alsoembodiments of the invention. In addition to methods of making theembodied nucleic acids and peptides, other embodiments include methodsof making vaccine compositions that can be used to treat or prevent HCVinfection. Some methods are practiced, for example, by mixing anadjuvant with a peptide or nucleic acid antigen (e.g., an HCV peptide orHCV nucleic acid, as described above so as to formulate a singlecomposition (e.g., a vaccine composition). Preferred methods involve themixing of ribavirin with an HCV gene or antigen disclosed herein.

Preferred methods of using the compositions described herein involveproviding an animal in need of a potent immune response to HCV with asufficient amount of one or more of the nucleic acid or peptideembodiments described herein. By one approach, for example, an animal inneed of potent immune response to HCV (e.g., an animal at risk oralready infected with HCV) is identified and said animal is provided anamount of NS3/4A (SEQ. ID. NO.: 2), a mutant NS3/4A (SEQ. ID. NOs.:3-13), a fragment thereof (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acidencoding said molecules that is effective to enhance or facilitate animmune response to the hepatitis viral antigen. Additional methods arepracticed by identifying an animal in need of a potent immune responseto HCV and providing said animal a composition comprising a peptidecomprising an antigen or epitope present on SEQ. ID. NOs.: 2-27 or anucleic acid encoding said peptide. Particularly preferred methodsinvolve the identification of an animal in need of an potent immuneresponse to HCV and providing said animal a composition comprising anamount of HCV antigen (e.g., NS3/4A (SEQ. ID. NO.: 2)), mutant NS3/4A(SEQ. ID. NOs.: 3-13), a fragment thereof at least 3, 4-10, 10-20,20-30, 30-50 amino acids in length (e.g., SEQ. ID. NOs.: 14-26) or anucleic acid encoding one or more of these molecules) that is sufficientto enhance or facilitate an immune response to said antigen. In someembodiments, the composition described above also contains an amount ofribavirin that provides an adjuvant effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the antibody titer in H-2^(d) mice against NS3as a function of time after the first immunization. Diamonds denoteantibody titer in mice immunized with NS3/4A-pVAX and squares denoteantibody titer in mice immunized with NS3-pVAX.

FIG. 2A is a graph showing the percentage of specific CTL-mediated lysisof SP2/0 target cells as a function of the effector to target ratio.Phosphate Buffered Saline (PBS) was used as a control immunogen.

FIG. 2B Is a graph showing the percentage specific CTL-mediated lysis ofSP2/0 target cells as a function of the effector to target ratio.Plasmid NS3/4A-pVAX was used as the immunogen.

FIG. 3 is a graph showing the humoral response to 10 and 100 μgrecombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), asdetermined by mean end point titres, when a single dose of 1 mg ofribavirin was co-administered.

FIG. 4 is a graph showing the humoral response to 20 μg recombinantHepatitis C virus (HCV) non structural 3 protein (NS3), as determined bymean end point titres, when a single dose of 0. 1, 1.0, or 10 mg ofribavirin was co-administered.

FIG. 5 is a graph showing the effects of a single dose of 1 mg ribavirinon NS3-specific lymph node proliferative responses, as determined by invitro recall responses.

DETAILED DESCRIPTION OF THE INVENTION

A novel nucleic acid and protein corresponding to the NS3/4A domain ofHCV was cloned from a patient infected with HCV (SEQ. ID. NO.: 1). AGenebank search revealed that the cloned sequence had the greatesthomology to HCV sequences but was only 93% homologous to the closest HCVrelative (accession no AJ 278830). This novel peptide (SEQ. ID. NO.: 2)and fragments thereof at least 3-20 amino acids in length (e.g., 3, 4,6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14and 15), nucleic acids encoding these molecules, vectors having saidnucleic acids, and cells having said vectors, nucleic acids, or peptidesare embodiments of the invention.

Mutants of the novel NS3/4A peptide were created. It was discovered thattruncated mutants (e.g., SEQ. ID. NOs.: 12 and 13) and mutants that lacka proteolytic cleavage site (SEQ. ID. NOs.: 3-11), are highlyimmunogenic. These novel peptides (SEQ. ID. NOs.: 3-13) and fragmentsthereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12,15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 16-26), nucleicacids encoding these molecules, vectors having said nucleic acids, andcells having said vectors, nucleic acids, or peptides are alsoembodiments of the invention.

The peptides and nucleic acids described above are useful as immunogens,which can be administered alone or in conjunction with an adjuvant.Preferred embodiments include compositions that comprise one or more ofthe nucleic acids and/or peptides described above and an adjuvant. Thatis, some of the vaccine embodiments described herein comprise anadjuvant and the novel NS3/4A peptide (SEQ. ID. NO.: 2) or a fragmentthereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12,15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15) or anucleic acid encoding one or more of these molecules. Additional vaccineembodiments comprise an adjuvant and one or more of the NS3/4A mutantpeptides (SEQ. ID. NOs.: 3-13) or a fragment thereof at least 3-20 aminoacids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids inlength) (e.g., SEQ. ID. NOs.: 16-26) or a nucleic acid encoding one ormore of these molecules.

It was also discovered that compositions comprising ribavirin and anantigen (e.g., a molecule containing an epitope of a pathogen such as avirus, bacteria, mold, yeast, parasite) enhance and/or facilitate ananimal's immune response to the antigen. That is, it was discovered thatribavirin is a very effective “adjuvant,” which for the purposes of thisdisclosure, refers to a material that has the ability to enhance orfacilitate an immune response to a particular antigen. The adjuvantactivity of ribavirin was manifested by a significant increase inimmune-mediated protection against the antigen, an increase in the titerof antibody raised to the antigen, and an increase in proliferative Tcell responses.

Accordingly, compositions (e.g., vaccines and other medicaments) thatcomprise ribavirin and one or more of the peptides or nucleic acidsdescribed herein are embodiments. These compositions can vary accordingto the amount of ribavirin, the form of ribavirin, as well as thesequence of the HCV nucleic acid or peptide.

Also embodied are methods of making and using the compositions above.Some methods involve the making of nucleic acids encoding NS3/4A, mutantNS34A, fragments thereof at least 9-30 consecutive nucleotides in length(e.g., 9, 12, 15, 18, 21, 24, 27, or 30 consecutive nucleotides inlength), peptides corresponding to said nucleic acids, constructscomprising said nucleic acids, and cells containing said compositions.Preferred methods, however, concern the making of vaccine compositionscomprising the newly discovered NS3/4A fragment or an NS3/4A mutant(e.g., a truncated mutant or a mutant lacking a proteolytic cleavagesite), or a fragment thereof of at least three amino acids in length ora nucleic acid encoding one or more of these molecules. Preferredfragments for use with the methods described herein include SEQ. ID.NOs.: 12-27. The compositions described above can be made by providingan adjuvant (e.g., ribavirin), providing an HCV antigen (e.g., a peptidecomprising an HCV antigen such as (SEQ. ID. NOs.: 2-11) or a fragmentthereof such as, SEQ. ID. NOs.: 12-26 or a nucleic acid encoding one ormore of said peptides), and mixing said ribavirin and said HCV antigenso as to formulate a composition that can be used to enhance orfacilitate an immune response in a subject to said antigen.

Methods of enhancing or facilitating the immune response of an animal,including humans, to an antigen are also desired. Such methods can bepracticed, for example, by identifying an animal in need of a potentimmune response to HCV and providing said animal a compositioncomprising one or more of the nucleic acids or peptides above and anamount of adjuvant (e.g., ribavirin) that is effective to enhance orfacilitate an immune response to the antigen/epitope. In someembodiments, the antigen and the adjuvant are administered separately,instead of in a single mixture. Preferably, in this instance, theribavirin is administered a short time before or a short time afteradmininstering the antigen. Preferred methods involve providing theanimal in need with ribavirin and NS3/4A (e.g., SEQ. ID. NO.: 2), amutant NS3/4A (e.g., SEQ. ID. NOs.: 3-13), a fragment thereof of atleast 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20amino acids in length) (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acidencoding said molecules.

Other embodiments of the invention concern methods of treating andpreventing HCV infection. By one approach, an immunogen comprising oneor more of the HCV nucleic acids or peptides described herein are usedto prepare a medicament for the treatment and/or prevention of HCVinfection. By another approach, an individual in need of a medicamentthat prevents and/or treats HCV infection is identified and saidindividual is provided a medicament comprising ribavirin and an HCVantigen such as NS3/4A (e.g., SEQ. ID. NO.: 2) or a mutant NS3/4A (e.g.,SEQ. ID. NOs.: 3-13), a fragment thereof of at least 3-20 amino acids inlength (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g.,SEQ. ID. NOs.: 14-26) or a nucleic acid encoding one or more of thesemolecules. The section below discusses the discovery of the novelNS3/4A, the creation of NS3/4A mutants, and the characterization ofthese nucleic acids and peptides corresponding thereto.

NS3/4A and NS3/4A Mutants

A novel nucleic acid and protein corresponding to the NS3/4A domain ofHCV was cloned from a patient infected with HCV (SEQ. ID. NOs.: 1 and2). A Genebank search revealed that the cloned sequence had the greatesthomology to HCV sequences but was only 93% homologous to the closest HCVrelative (accession no AJ 278830). A truncated mutant of the novelNS3/4A peptide and NS3/4A mutants, which lack a proteolytic cleavagesite, were also created. It was discovered that these novel peptides andnucleic acids encoding said peptides were potent immunogens that can bemixed with adjuvants (e.g., ribavirin) so as to make a composition thatprovides a recipient with a potent immune response to HCV. The cloningof the novel NS3/4A domain and the creation of the various NS3/4Amutants are described in the following example.

EXAMPLE 1

The NS3/4A sequence was amplified from the serum of an HCV-infectedpatient (HCV genotype 1a) using the Polymerase Chain Reaction (PCR).Total RNA was extracted from serum, and cDNA synthesis and PCR wereperformed according to standard protocols (Chen M et al., J. Med. Virol.43:223-226 (1995), herein expressly incorporated by reference in itsentirety). The cDNA synthesis was initiated using the antisense primer“NS4KR” (5′-CCG TCT AGA TCA GCA CTC TTC CAT TTC ATC-3′ (SEQ. ID. NO.:28)). From this cDNA, a 2079 base pair DNA fragment of HCV,corresponding to amino acids 1007 to 1711, which encompasses the NS3 andNS4A genes, was amplified. A high fidelity polymerase (Expand HighFidelity PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the“NS3KF” primer (5′-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3′ (SEQ. ID.NO.: 29) and the NS4KR primer. The NS3KF primer contained a EcoRIrestriction enzyme cleavage site and a start codon and the primer NS4KRcontained a XbaI restriction enzyme cleavage site and a stop codon.

The amplified fragment was then sequenced (SEQ. ID. NO.: 1). Sequencecomparison analysis revealed that the gene fragment was indeed amplifiedfrom a viral strain of genotype 1a. A computerized BLAST search againstthe Genbank database using the NCBI website revealed that the closestHCV homologue was 93% identical in nucleotide sequence.

The amplified DNA fragment was then digested with EcoRI and XbaI, andwas inserted into a pcDNA3.1/His plasmid (Invitrogen) digested with thesame enzymes. The NS3/4A-pcDNA3.1 plasmid was then digested with EcoRIand Xba I and the insert was purified using the QiaQuick kit (Qiagen,Hamburg, Germany) and was ligated to a EcoRI/Xba I digested pVAX vector(Invitrogen) so as to generate the NS3/4A-pVAX plasmid.

The rNS3 truncated mutant was obtained by deleting NS4A sequence fromthe NS3/4A DNA. Accordingly, the NS3 gene sequence of NS3/4A-pVAX wasPCR amplified using the primers NS3KF and 3′NotI (5′-CCA CGC GGC CGC GACGAC CTA CAG-3′ (SEQ. ID. NO.: 30)) containing EcoRI and Not Irestriction sites, respectively. The NS3 fragment (1850 bp) was thenligated to a EcoRI and Not I digested pVAX plasmid to generate theNS3-pVAX vector. Plasmids were grown in BL21 E.coli cells. The plasmidswere sequenced and were verified by restriction cleavage and the resultswere as to be expected based on the original sequence.

TABLE 1 describes the sequence of the proteolytic clevage site ofNS3/4A, referred to as the breakpoint between NS3 and NS4A. Thiswild-type breakpoint sequence was mutated in many different ways so asto generate several different NS3/4A breakpoint mutants. TABLE 1 alsoidentifies these mutant breakponit sequences. The fragments listed inTABLE 1 are preferred immunogens that can be incorporated with orwithout an adjuvant (e.g., ribavirin) into a composition foradministration to an animal so as to induce an immune response in saidanimal to HCV.

To change the proteolytic cleavage site between NS3 and NS4A, theNS3/4A-pVAX plasmid was mutagenized using the QUICKCHANGE™ mutagenesiskit (Stratagene), following the manufacturer's recommendations. Togenerate the “TPT” mutation, for example, the plasmid was amplifiedusing the primers 5′-CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3′ (SEQ. ID. NO.:31) and 5′-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3′ (SEQ. ID. NO.: 32)resulting in NS3/4A-TPT-pVAX. To generate the “RGT” mutation, forexample, the plasmid was amplified using the primers5′-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3′ (SEQ. ID. NO.: 33) and5′-ACCGAGCACCCAGGTACC-GCGGACGACCTCCAG-3′ (SEQ. ID. NO.: 34) resulting inNS3/4A-RGT-pVAX.

All mutagenized constructs were sequenced to verify that the mutationshad been correctly made. Plasmids were grown in competent BL21 E. coli.The plasmid DNA used for in vivo injection was purified using Qiagen DNApurification columns, according to the manufacturers instructions(Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmidDNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech,Uppsala, Sweden) and the purified DNA was dissolved in sterile phosphatebuffer saline (PBS) at concentrations of 1 mg/ml.

TABLE 1 Plasmid Deduced amino acid sequence (SEQ. ID. NO.: 14)*NS3/4A-pVAX TKYMTCMSADLEVVTSTWVLVGGVL (SEQ. ID. NO.: 16)NS3/4A-TGT-pVAX TKYMTCMSADLEVVTGTWVLVGGVL (SEQ. ID. NO.: 17)NS3/4A-RGT-pVAX TKYMTCMSADLEVVRGTWVLVGGVL (SEQ. ID. NO.: 18)NS3/4A-TPT-pVAX TKYMTCMSADLEVVTPTWVLVGGVL (SEQ. ID. NO.: 19)NS3/4A-RPT-pVAX TKYMTCMSADLEVVRPTWVLVGGVL (SEQ. ID. NO.: 20)NS3/4A-RPA-pVAX TKYMTCMSADLEVVRPAWVLVGGVL (SEQ. ID. NO.: 21)NS3/4A-CST-pVAX TKYMTCMSADLEVVCSTWVLVGGVL (SEQ. ID. NO.: 22)NS3/4A-CCST-pVAX TKYMTCMSADLEVCCSTWVLVGGVL (SEQ. ID. NO.: 23)NS3/4A-SSST-pVAX TKYMTCMSADLEVSSSTWVLVGGVL (SEQ. ID. NO.: 24)NS3/4A-SSSSCST-pVAX  TKYMTCMSADSSSSCSTWVLVGGVL (SEQ. ID. NO.: 25)NS3A/4A-VVVVTST-pVAX    TKYMTCMSADVVVVTSTWVLVGGVL (SEQ. ID. NO.: 27)NS5-pVAX       ASEDVVCCSMSYTWTG (SEQ. ID. NO.: 26) NS5A/B-pVAX      SSEDVVCCSMWVLVGGVL *The wild type sequence for the NS3/4A fragmentis NS3/4A-pVAX. The NS3/4A breakpoint is identified by underline,wherein the P1 position corresponds to the first Thr (T) and theP1′ position corresponds to the next following amino acid theNS3/4A-pVAX sequence. In the wild type NS3/4A sequence the NS3 proteasecleaves between the P1 and P1′ positions.

The nucleic acid embodiments include nucleotides encoding the HCVpeptides described herein (SEQ. ID. NOs.: 2-11) or fragments thereof atleast 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15). Someembodiments for example, include genomic DNA, RNA, and cDNA encodingthese HCV peptides. The HCV nucleotide embodiments not only include theDNA sequences shown in the sequence listing (e.g., SEQ. ID. NO.: 1) butalso include nucleotide sequences encoding the amino acid sequencesshown in the sequence listing (e.g., SEQ. ID. NOs.: 2-11) and anynucleotide sequence that hybridizes to the DNA sequences shown in thesequence listing under stringent conditions (e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7.0% sodium dodecyl sulfate (SDS), 1mM EDTA at 50° C.) and washing in 0.2×SSC/0.2% SDS at 50° C. and anynucleotide sequence that hybridizes to the DNA sequences that encode anamino acid sequence provided in the sequence listing (SEQ. ID. NOs.:2-11) under less stringent conditions (e.g., hybridization in 0.5 MNaHPO₄, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37° C. andwashing in 0.2×SSC/0.2% SDS at 37° C.).

The nucleic acid embodiments of the invention also include fragments,modifications, derivatives, and variants of the sequences describedabove. Desired embodiments, for example, include nucleic acids having atleast 12 consecutive bases of one of the novel HCV sequences or asequence complementary thereto and preferred fragments include at least12 consecutive bases of a nucleic acid encoding the NS3/4A molecule ofSEQ. ID. NO.: 2 or a mutant NS3/4A molecule of SEQ. ID. NOs.: 3-13 or asequence complementary thereto.

In this regard, the nucleic acid embodiments described herein can havefrom 12 to approximately 2079 consecutive nucleotides. Some DNAfragments, for example, include nucleic acids having at least 12-15,15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000, 1000-1500, or1500-2079 consecutive nucleotides of SEQ. ID. NO.: 1 or a complementthereof. These nucleic acid embodiments can also be altered bysubstitution, addition, or deletion so long as the alteration does notsignificantly affect the structure or function (e.g., ability to serveas an immunogen) of the HCV nucleic acid. Due to the degeneracy ofnucleotide coding sequences, for example, other DNA sequences thatencode substantially the same HCV amino acid sequence as depicted inSEQ. ID. NOs.: 2-13 can be used in some embodiments. These include, butare not limited to, nucleic acid sequences encoding all or portions ofHCV peptides (SEQ. ID. NOs.: 2-13) or nucleic acids that complement allor part of this sequence that have been altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change, or afunctionally non-equivalent amino acid residue within the sequence, thusproducing a detectable change. Accordingly, the nucleic acid embodimentsof the invention are said to be consisting essentially of nucleic acidsencoding any one of SEQ. ID. NOs.: 2-27 in light of the modificationsabove.

By using the nucleic acid sequences described above, probes thatcomplement these molecules can be designed and manufactured byoligonucleotide synthesis. Desirable probes comprise a nucleic acidsequence of (SEQ. ID. NO.: 1) that is unique to this HCV isolate. Theseprobes can be used to screen cDNA from patients so as to isolate naturalsources of HCV, some of which may be novel HCV sequences in themselves.Screening can be by filter hybridization or by PCR, for example. Byfilter hybridization, the labeled probe preferably contains at least15-30 base pairs of the nucleic acid sequence of (SEQ. ID. NO.: 1) thatis unique to to this NS3/4A peptide. The hybridization washingconditions used are preferably of a medium to high stringency. Thehybridization can be performed in 0.5M NaHPO₄, 7.0% sodium dodecylsulfate (SDS), 1 mM EDTA at 42° C. overnight and washing can beperformed in 0.2×SSC/0.2% SDS at 42° C. For guidance regarding suchconditions see, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al.,1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. both of which are hereinexpressly incorporated by reference.

HCV nucleic acids can also be isolated from patients infected with HCVusing the nucleic acids described herein. (See also Example 1).Accordingly, RNA obtained from a patient infected with HCV is reversetranscribed and the resultant cDNA is amplified using PCR or anotheramplification technique. The primers are preferably obtained from theNS3/4A sequence (SEQ. ID. NO.: 1).

For a review of PCR technology, see Molecular Cloning to GeneticEngineering White, B. A. Ed. in Methods in Molecular Biology 67: HumanaPress, Totowa (1997), the disclosure of which is incorporated herein byreference in its entirety and the publication entitled “PCR Methods andApplications” (1991, Cold Spring Harbor Laboratory Press), thedisclosure of which is incorporated herein by reference in its entirety.For amplification of mRNAs, it is within the scope of the invention toreverse transcribe mRNA into cDNA followed by PCR (RT-PCR); or, to use asingle enzyme for both steps as described in U.S. Pat. No. 5,322,770,the disclosure of which is incorporated herein by reference in itsentirety. Another technique involves the use of Reverse TranscriptaseAsymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described byMarshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994), thedisclosure of which is incorporated herein by reference in its entirety.

Briefly, RNA is isolated, following standard procedures. A reversetranscription reaction is performed on the RNA using an oligonucleotideprimer specific for the most 5′ end of the amplified fragment as aprimer of first strand synthesis. The resulting RNA/DNA hybrid is then“tailed” with guanines using a standard terminal transferase reaction.The hybrid is then digested with RNAse H, and second strand synthesis isprimed with a poly-C primer. Thus, cDNA sequences upstream of theamplified fragment are easily isolated. For a review of cloningstrategies which can be used, see e.g., Sambrook et al., 1989, supra.

In each of these amplification procedures, primers on either side of thesequence to be amplified are added to a suitably prepared nucleic acidsample along with dNTPs and a thermostable polymerase, such as Taqpolymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in thesample is denatured and the primers are specifically hybridized tocomplementary nucleic acid sequences in the sample. The hybridizedprimers are then extended. Thereafter, another cycle of denaturation,hybridization, and extension is initiated. The cycles are repeatedmultiple times to produce an amplified fragment containing the nucleicacid sequence between the primer sites. PCR has further been describedin several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and4,965,188, the disclosures of which are incorporated herein by referencein their entirety.

The primers are selected to be substantially complementary to a portionof the nucleic acid sequence of (SEQ. ID. NO.: 1) that is unique to thisNS3/4A molecule, thereby allowing the sequences between the primers tobe amplified. Preferably, primers are at least 16-20, 20-25, or 25-30nucleotides in length. The formation of stable hybrids depends on themelting temperature (Tm) of the DNA. The Tm depends on the length of theprimer, the ionic strength of the solution and the G+C content. Thehigher the G+C content of the primer, the higher is the meltingtemperature because G:C pairs are held by three H bonds whereas A:Tpairs have only two. The G+C content of the amplification primersdescribed herein preferably range between 10 and 75%, more preferablybetween 35 and 60%, and most preferably between 40 and 55%. Theappropriate length for primers under a particular set of assayconditions can be empirically determined by one of skill in the art.

The spacing of the primers relates to the length of the segment to beamplified. In the context of the embodiments described herein, amplifiedsegments carrying nucleic acid sequence encoding HCV peptides can rangein size from at least about 25 bp to the entire length of the HCVgenome. Amplification fragments from 25-1000 bp are typical, fragmentsfrom 50-1000 bp are preferred and fragments from 100-600 bp are highlypreferred. It will be appreciated that amplification primers can be ofany sequence that allows for specific amplification of the NS3/4A regionand can, for example, include modifications such as restriction sites tofacilitate cloning.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an HCV peptide. The PCRfragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library. Additionally, anexpression library can be constructed utilizing cDNA synthesized from,for example, RNA isolated from an infected patient. In this manner, HCVgeneproducts can be isolated using standard antibody screeningtechniques in conjunction with antibodies raised against the HCV geneproduct. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor., herein expressly incorporated by referencein its entirety)

Embodiments of the invention also include (a) DNA vectors that containany of the foregoing nucleic acid sequence and/or their complements(i.e., antisense); (b) DNA expression vectors that contain any of theforegoing nucleic acid sequences operatively associated with aregulatory element that directs the expression of the nucleic acid; and(c) genetically engineered host cells that contain any of the foregoingnucleic acid sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences in the host cell.These recombinant constructs are capable of replicating autonomously ina host cell. Alternatively, the recombinant constructs can becomeintegrated into the chromosomal DNA of a host cell. Such recombinantpolynucleotides typically comprise an HCV genomic or cDNA polynucleotideof semi-synthetic or synthetic origin by virtue of human manipulation.Therefore, recombinant nucleic acids comprising these sequences andcomplements thereof that are not naturally occurring are provided.

Although nucleic acids encoding an HCV peptide or nucleic acids havingsequences that complement an HCV gene as they appear in nature can beemployed, they will often be altered, e.g., by deletion, substitution,or insertion and can be accompanied by sequence not present in humans.As used herein, regulatory elements include, but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include, but are not limited to,the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus, the lac system, the trp system, the TACsystem, the TRC system, the major operator and promoter regions of phageA, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters of the yeast -mating factors.

In addition, recombinant HCV peptide-encoding nucleic acid sequences andtheir complementary sequences can be engineered so as to modify theirprocessing or expression. For example, and not by way of limitation, theHCV nucleic acids described herein can be combined with a promotersequence and/or ribosome binding site, or a signal sequence can beinserted upstream of HCV peptide-encoding sequences so as to permitsecretion of the peptide and thereby facilitate harvesting orbioavailability. Additionally, a given HCV nucleic acid can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction sites or destroy preexisting ones, or tofacilitate further in vitro modification. (See Example 1). Any techniquefor mutagenesis known in the art can be used, including but not limitedto, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol.Chem., 253:6551 (1978), herein incorporated by reference in itsentirety).

Further, nucleic acids encoding other proteins or domains of otherproteins can be joined to nucleic acids encoding an HCV peptide so as tocreate a fusion protein. Nucleotides encoding fusion proteins caninclude, but are not limited to, a full length NS3/4A sequence (SEQ. ID.NO.: 2), mutant NS3/4A sequences (e.g., SEQ. ID. NOs.: 3-11) or apeptide fragment of an NS3/4A sequence fused to an unrelated protein orpeptide, such as for example, poly histidine, hemagglutinin, an enzyme,fluorescent protein, or luminescent protein, as discussed below.Surprisingly, it was discovered that the NS3/4A-pVAX was significantlymore immunogenic than NS3-pVAX vectors when injected into animmunocompetent mammal. The example below describes these experiments ingreater detail.

EXAMPLE 2

To determine whether a humoral immune response was elicited by theNS3-pVAX and NS3/4A-pVAX vectors, the expression constructs described inExample 1 were purified using the Qiagen DNA purification system,according to the manufacturer's instructions and the purified DNAvectors were used to immunize groups of four to ten Balb/c mice. Theplasmids were injected directly into regenerating tibialis anterior (TA)muscles as previously described (Davis et al., Human Gene Therapy4(6):733 (1993), herein expressly incorporated by reference). In brief,mice were injected intramuscularly with 50 μl/TA of 0.01 mM cardiotoxin(Latoxan, Rosans, France) in 0.9% sterile NaCl. Five days later, each TAmuscle was injected with 50 μl PBS containing either rNS3 or DNA.

Inbred mouse strains C57/BL6 (H-2b) Balb/C (H-2d), and CBA (H-2k) wereobtained from the breeding facility at Möllegard Denmark, Charles RiverUppsala, Sweden, or B&K Sollentuna Sweden. All mice were female and wereused at 4-8 weeks of age. For monitoring of humoral responses, all micereceived a booster injection of 50 μl /TA of plasmid DNA every fourthweek. In addition, some mice were given recombinant NS3 (rNS3) protein,which was purified, as described herein. The mice receiving rNS3 wereimmunized no more than twice. All mice were bled twice a month.

Enzyme immunosorbent assays (EIAs) were used to detect the presence ofmurine NS3 antibodies. These assays were performed essentially asdescribed (Chen et al., Hepatology 28(1): 219 (1998)). Briefly, rNS3 waspassively adsorbed overnight at 4° C. to 96-well microtiter plates(Nunc, Copenhagen, Denmark) at 1 μg/ml in 50 mM sodium carbonate buffer(pH 9.6). The plates were then blocked by incubation with dilutionbuffer containing PBS, 2% goat serum, and 1% bovine serum albumin forone hour at 37° C. Serial dilutions of mouse sera starting at 1:60 werethen incubated on the plates for one hour. Bound murine serum antibodieswere detected by an alkaline phosphatase conjugated goat anti-mouse IgG(Sigma Cell Products, Saint Louis, Mo.) followed by addition of thesubstrate pNPP (1 tablet/5 ml of 1M Diethanol amine buffer with 0.5 mMMgCl₂). The reaction was stopped by addition of 1M NaOH and absorbencywas read at 405 nm.

After four weeks, four out of five mice immunized with NS3/4A-pVAX haddeveloped NS3 antibodies, whereas one out of five immunized withNS3-pVAX had developed antibodies (FIG. 1). After six weeks, four out offive mice immunized with NS3/4A-pVAX had developed high levels (>10⁴) ofNS3 antibodies (mean levels 10800±4830) and one had a titer of 2160.Although all mice immunized with NS3-pVAX developed NS3 antibodies, noneof them developed levels as high as that produced by the NS3/4A-pVAXconstruct (mean levels 1800±805). The antibody levels elicited by theNS3/4A fusion construct were significantly higher than those induced byNS3-pVAX at six weeks (mean ranks 7.6 v.s 3.4, p<0.05, Mann-Whitney ranksum test, and p<0.01, Students t-test). Thus, immunization with eitherNS3-pVAX or NS3/4A-pVAX resulted in the production of anti-NS3antibodies, but the NS3/4A fusion gene was a more potent immunogen. Theexample below describes experiments that were performed to determine ifmutant NS3/4A peptides, which lack a proteolytic clevage site, couldelicit a potent immune response.

EXAMPLE 3

To test if the enhanced immunogenicity of NS3/4A could be solelyattributed to the presence of NS4A, or if the NS3/4A fusion protein inaddition had to be cleaved at the NS3/4A junction, new experiments wereperformed. In a first experiment, the immunogenicity of the NS3-pVAX,NS3/4A-pVAX, and mutant NS3/4A constructs were compared in Balb/c mice.Mice were immunized on week 0 as described above, and, after two weeks,all mice were bled and the presence of antibodies to NS3 at a serumdilution of 1:60 was determined (TABLE 2). Mice were bled again on week4. As shown in TABLE 2, all the constructs induced an immune reponse;the mutant constructs, for example, the NS3/4A-TGT-pVAX vector wascomparable to the NS3-pVAX vector (4/10 vs. 0/10; NS, Fisher's exacttest). The NS3/4A-pVAX vector, however, continued to be the most potentimmunogen. Thus, all of the HCV constructs that were introduced intomice were capable of eliciting an immune response against NS3, however,the NS4A sequence and a functional proteolytic cleavage site between theNS3 and NS4A sequences provided for a more potent immune response.

TABLE 2 No. of antibody responders to the respective immunogen after one100 μg i.m immunization mutant example Weeks from 1^(st) wild-typeNS3/4A-TGT- immunization NS3-pVAX NS3/4A-pVAX pVAX 2 0/10 17/20 4/10 40/10 20/20 10/10  (<60) (2415 ± 3715) (390 ± 639) 55% > 10³ 50% > 10²10% > 10⁴ 10% > 10³

During the chronic phase of infection, HCV replicates in hepatocytes,and spreads within the liver. A major factor in combating chronic andpersistent viral infections is the cell-mediated immune defense system.CD4+ and CD8+ lymphocytes infiltrate the liver during the chronic phaseof HCV infection, but they are incapable of clearing the virus orpreventing liver damage. In addition, persistent HCV infection isassociated with the onset of hepatocellular carcinoma (HCC). Theexamples below describe experiments that were performed to determinewhether the NS3 and NS3/4A construct were capable of eliciting a T-cellmediated immune response against NS3.

EXAMPLE 4

To study whether the constructs described above were capable ofeliciting a cell-mediated response against NS3, an in vivo tumor growthassay was perfomed. To this end, an SP2/0 tumor cell line stablytransfected with the NS3/4A gene was made. The pcDNA3.1 plasmidcontaining the NS3/4A gene was linearized by BglII digestion. A total of5 μg linearized plasmid DNA was mixed with 60 μg transfection reagent(Superfect, Qiagen, Germany) and the mixture was added to a 50%confluent layer of SP2/0 cells in a 35 mm dish. The transfected SP2/0cells (NS3/4A-SP2/0) were grown for 14 days in the presence of 800 μg/mlgeneticin and individual clones were isolated. A stableNS3/4A-expressing SP2/0 clone was identified using PCR and RTPCR. Thecloned cell line was maintained in DMEM containing 10% fetal bovineserum, L-glutamine, and penicillin-streptomycin.

The in vivo growth kinetics of the SP2/0 and the NS3/4A-SP2/0 cell lineswere then evaluated in Balb/c mice. Mice were injected subcutaneouslywith 2×10⁶ tumor cells in the right flank. Each day the size of thetumor was determined through the skin. The growth kinetics of the twocell lines was comparable. The mean tumor sizes did not differ betweenthe two cell lines at any time point, for example. (See TABLE 3). Theexample below describes experiments that were performed to determinewhether mice immunized with the NS3/4A constructs had developed a T-cellresponse against NS3.

TABLE 3 Mouse Tumor Maximum in vivo tumor size at indicated time pointID cell line 5 6 7 8 11 12 13 14 15 1 SP2/0 1.6 2.5 4.5 6.0 10.0 10.511.0 12.0 12.0 2 SP2/0 1.0 1.0 2.0 3.0 7.5 7.5 8.0 11.5 11.5 3 SP2/0 2.05.0 7.5 8.0 11.0 11.5 12.0 12.0 13.0 4 SP2/0 4.0 7.0 8.0 10.0 13.0 15.016.5 16.5 17.0 5 SP2/0 1.0 1.0 3.0 4.0 5.0 6.0 6.0 6.0 7.0 Group mean1.92 3.3 5.0 6.2 9.3 10.1 10.7 11.6 12.1 6 NS3/4A-SP2/0 1.0 2.0 3.0 3.54.0 5.5 6.0 7.0 8.0 7 NS3/4A-SP2/0 2.0 2.5 3.0 5.0 7.0 9.0 9.5 9.5 11.08 NS3/4A-SP2/0 1.0 2.0 3.5 3.5 9.5 11.0 12.0 14.0 14.0 9 NS3/4A-SP2/01.0 1.0 2.0 6.0 11.5 13.0 14.5 16.0 18.0 10  NS3/4A-SP2/0 3.5 6.0 7.010.5 15.0 15.0 15.0 15.5 20.0 Group mean 1.7 2.7 3.7 5.7 9.4 10.7 11.412.4 14.2 p-value of student's t- 0.7736 0.6918 0.4027 0.7903 0.96700.7986 0.7927 0.7508 0.4623 test comparison between group means

EXAMPLE 5

To examine whether a T-cell response is elicited by the NS3/4Aimmunization, the capacity of an immunized mouse's immune defense systemto attack the NS3-expressing tumor cell line was assayed. The protocolfor testing for in vivo inhibition of tumor growth of the SP2/0 myelomacell line in Balb/c mice has been described in detail previously (Enckeet al., J. Immunol. 161:4917 (1998), herein expressly incorporated byreference in its entirety). Inhibition of tumor growth in this model isdependent on the priming of cytotoxic T lymphocytes (CTLs). Briefly,groups of ten mice were immunized i.m. five times with one monthintervals with either 100 μg NS3-pVAX or 100 μg NS3/4A-pVAX. Two weeksafter the last immunization 2×10⁶ SP2/0 or NS3/4A-SP2/0 cells wereinjected into the right flank of each mouse. Two weeks later the micewere sacrificed and the maximum tumor sizes were measured. There was nodifference between the mean SP2/0 and NS3/4A-SP2/0 tumor sizes in theNS3-pVAX immunized mice (See TABLE 4).

TABLE 4 Maximum Mouse Dose Tumor cell Tumor tumor size ID Immunogen (μg)line growth (mm) 1 NS3-pVAX 100 SP2/0 Yes 5 2 NS3-pVAX 100 SP2/0 Yes 153 NS3-pVAX 100 SP2/0 No — 4 NS3-pVAX 100 SP2/0 Yes 6 5 NS3-pVAX 100SP2/0 Yes 13 Group total 4/5 9.75 ± 4.992 6 NS3-pVAX 100 NS3/4A-SP2/0Yes 9 7 NS3-pVAX 100 NS3/4A-SP2/0 Yes 8 8 NS3-pVAX 100 NS3/4A-SP2/0 Yes7 9 NS3-pVAX 100 NS3/4A-SP2/0 No — 10  NS3-pVAX 100 NS3/4A-SP2/0 No —3/5 8.00 ± 1.00  Note: Statistical analysis (StatView): Student's t-teston maximum tumor size. P-values < 0.05 are considered significant.Unpaired t-test for Max diam

-   Grouping Variable: Column 1-   Hypothesized Difference=0-   Row exclusion: NS3DNA-Tumor-001213

Mean Diff. DF t-Value P-Value NS3-sp2, NS3-spNS3 1.750 5 0.58 0.584Group Info for Max diam

-   Grouping Variable: Column 1-   Row exclusion: NS3DNA-Tumor-001213

Count Mean Variance Std. Dev. Std. Err NS3-sp2 4 9.750 24.917 4.9922.496 NS3-spNS3 3 8.000 1.000 1.000 0.57In the next set of experiments, the inhibition of SP2/0 or NS3/4A-SP2/0tumor growth was evaluated in NS3/4A-pVAX immunized Balb/c mice. In miceimmunized with the NS3/4A-pVAX plasmid the growth of NS3/4A-SP2/0 tumorcells was significantly inhibited as compared to growth of thenon-transfected SP2/0 cells. (See TABLE 5). Thus, NS3/4A-pVAXimmunization elicits CTLs that inhibit growth of cells expressing NS3/4Ain vivo. The example below describes experiments that were performed toanalyze the efficiency of various NS3 containing compositions ineliciting a cell-mediated response to NS3.

TABLE 5 Maximum Mouse Dose Tumor cell Tumor tumor size ID Immunogen (μg)line growth (mm) 11 NS3/4A-pVAX 100 SP2/0 No — 12 NS3/4A-pVAX 100 SP2/0Yes 24 13 NS3/4A-pVAX 100 SP2/0 Yes 9 14 NS3/4A-pVAX 100 SP2/0 Yes 11 15NS3/4A-pVAX 100 SP2/0 Yes 25 4/5 17.25 ± 8.421 16 NS3/4A-pVAX 100NS3/4A- No — SP2/0 17 NS3/4A-pVAX 100 NS3/4A- Yes 9 SP2/0 18 NS3/4A-pVAX100 NS3/4A- Yes 7 SP2/0 19 NS3/4A-pVAX 100 NS3/4A- Yes 5 SP2/0 20NS3/4A-pVAX 100 NS3/4A- Yes 4 SP2/0 4/5  6.25 ± 2.217 Note: Statisticalanalysis (StatView): Student's t-test on maximum tumor size. P-values <0.05 are considered significant.Unpaired t-test for Max diam

-   Grouping Variable: Column 1-   Hypothesized Difference=0-   Row exclusion: NS3DNA-Tumor001213

Mean Diff. DF t-Value P-Value NS3/4-sp2, NS3/4-spNS3 11.000 6 2.5260.044Group Info for Max diam

-   Grouping Variable: Column 1-   Row exclusion: NS3DNA-Tumor-001213

Count Mean Variance Std. Dev. Std. Err NS3/4-sp2 4 17.250 70.917 8.4214.211 NS3/4-spNS3 4 6.250 4.917 2.217 1.109

EXAMPLE 6

To analyze whether administration of different NS3 containingcompositions affected the elicitation of a cell-mediated immuneresponse, mice were immunized with PBS, rNS3, irrelevant DNA or theNS3/4A construct, and tumor sizes were determined, as described above.Only the NS3/4A construct was able to elicit a T-cell responsesufficient to cause a statistically significant reduction in tumor size(See TABLE 6). The example below describes experiments that wereperformed to determine whether the reduction in tumor size can beattributed to the generation of NS3-specific T-lymphocytes.

TABLE 6 Maximum Dose Anti- Tumor tumor size Mouse ID Immunogen (μg)Tumor cell line NS3 growth (mm) 1 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 12.0 2NS3-pVAX 10 NS3/4A-SP2/0 <60 + 20.0 3 NS3-pVAX 10 NS3/4A-SP2/0 60 + 18.04 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 13.0 5 NS3-pVAX 10 NS3/4A-SP2/0 <60 +17.0 Group mean 60 5/5  16.0 ± 3.391 6 NS3-pVAX 100 NS3/4A-SP2/0 2160 +10.0 7 NS3-pVAX 100 NS3/4A-SP2/0 <60 − − 8 NS3-pVAX 100 NS3/4A-SP2/0 <60− − 9 NS3-pVAX 100 NS3/4A-SP2/0 360 − − 10 NS3-pVAX 100 NS3/4A-SP2/0<60 + 12.5 Group mean 1260 2/5 11.25 ± 1.768 11 NS3/4A-pVAX 10NS3/4A-SP2/0 <60 + 10.0 12 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 − − 13NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 − − 14 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 +13.0 15 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 13.5 Group mean <60 3/5 12.167± 1.893  16 NS3/4A-pVAX 100 NS3/4A-SP2/0 60 + 10.0 17 NS3/4A-pVAX 100NS3/4A-SP2/0 360 − − 18 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 8.0 19NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 12.0 20 NS3/4A-pVAX 100 NS3/4A-SP2/02160 + 7.0 Group mean 1380 4/5  9.25 ± 2.217 36 p17-pcDNA3 100NS3/4A-SP2/0 <60 + 20.0 37 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 7.0 38p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 11.0 39 p17-pcDNA3 100 NS3/4A-SP2/0<60 + 15.0 40 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 18.0 Group mean <60 5/514.20 ± 5.263 41 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 13.0 42 rNS3/CFA 20NS3/4A-SP2/0 >466560 − − 43 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 3.5 44rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 22.0 45 rNS3/CFA 20NS3/4A-SP2/0 >466560 + 17.0 Group mean 466560 4/5 17.333 ± 4.509  46 PBS— NS3/4A-SP2/0 <60 + 10.0 47 PBS — NS3/4A-SP2/0 <60 + 16.5 48 PBS —NS3/4A-SP2/0 60 + 15.0 49 PBS — NS3/4A-SP2/0 <60 + 21.0 50 PBS —NS3/4A-SP2/0 <60 + 15.0 51 PBS — NS3/4A-SP2/0 <60 − − Group mean 60 5/615.50 ± 3.937 Note: Statistical analysis (StatView): Student's t-test onmaximum tumor size. P-values < 0.05 are considered as significant.Unpaired t-test for Largest Tumor size

-   Grouping Variable: group-   Hypothesized Difference=0

Mean Diff. DF t-value P-Value p17-sp3-4, NS3-100-sp3-4 2.950 5 .739.4933 p17-sp3-4, NS3/4-10-sp3-4 2.033 6 .628 .5532 p17-sp3-4,NS3-10-sp3-4 −1.800 8 −.643 .5383 p17-sp3-4, NS3/4-100-sp3-4 4.950 71.742 .1250 p17-sp3-4, PBS-sp3-4 −1.300 8 −.442 .6700 p17-sp3-4,rNS3-sp3-4 −3.133 6 −.854 .4259 NS3-100-sp3-4, NS3/4-10-sp3-4 −.917 3−.542 .6254 NS3-100-sp3-4, NS3-10-sp3-4 −4.750 5 −1.811 .1299NS3-100-sp3-4, NS3/4-100-sp3-4 2.000 4 1.092 .3360 NS3-100-sp3-4,PBS-sp3-4 −4.250 5 −1.408 .2183 NS3-100-sp3-4, rNS3-sp3-4 −6.083 3−1.744 .1795 NS3/4-10-sp3-4, NS3-10-sp3-4 −3.833 6 −1.763 .1283NS3/4-10-sp3-4, NS3/4-100-sp3-4 2.917 5 1.824 .1277 NS3/4-10-sp3-4,PBS-sp3-4 −3.333 6 −1.344 .2274 NS3/4-10-sp3-4, rNS3-sp3-4 −5.167 4−1.830 .1412 NS3-10-sp3-4, NS3/4-100-sp3-4 6.750 7 3.416 .0112NS3-10-sp3-4, PBS-sp3-4 .500 8 .215 .8350 NS3-10-sp3-4, rNS3-sp3-4−1.333 6 −.480 .6480 NS3/4-100-sp3-4, PBS-sp3-4 −6.250 7 −2.814 .0260NS3/4-100-sp3-4, rNS3-sp3-4 −8.083 5 −3.179 .0246 PBS-sp3-4, rNS3-sp3-4−1.833 6 −.607 .5662

EXAMPLE 7

To determine whether NS3-specific T-cells were elicited by the NS3/4Aimmunizations, an in vitro T-cell mediated tumor cell lysis assay wasemployed. The assay has been described in detail previously (Sallberg etal., J. Virol. 71:5295 (1997), herein expressly incorporated byreference in its entirety). Briefly, groups of five Balb/c mice wereimmunized three times with 100 μg NS3/4A-pVAX i.m. Two weeks after thelast injection the mice were sacrificed and splenocytes were harvested.Re-stimulation cultures with 3×10⁶ splenocytes and 3×10⁶ NS3/4A-SP2/0cells were set. After five days, a standard Cr⁵¹-release assay wasperformed using NS3/4A-SP2/0 or SP2/0 cells as targets. Percent specificlysis was calculated as the ratio between lysis of NS3/4A-SP2/0 cellsand lysis of SP2/0 cells. Only mice immunized with NS3/4A-pVAX displayedspecific lysis over 10% in four out of five tested mice, using aneffector to target ratio of 20:1 (See FIGS. 2A and 2B). The sectionbelow describes several of the embodied HCV polypeptides in greaterdetail.

The nucleic acids encoding the HCV peptides, described above, can bemanipulated using conventional techniques in molecular biology so as tocreate recombinant constructs that express the HCV peptides. Theembodied HCV peptides or derivatives thereof, include but are notlimited to, those containing as a primary amino acid sequence all of theamino acid sequence substantially as depicted in the Sequence Listing(SEQ. ID. NOs.: 2-11) and fragments of SEQ. ID. NOs.: 2-11 at least fouramino acids in length (e.g., SEQ. ID. NOs.: 14-16) including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. Preferred fragments of a sequence of SEQ. ID. NOs.: 2-11 are atleast four amino acids and comprise amino acid sequence unique to thediscovered NS3/4A peptide or mutants thereof including altered sequencesin which functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. The HCVpeptides can be, for example, at least (12-704 amino acids in length(e.g., 12-15, 15-20, 20-25, 25-50, 50-100, 100-150, 150-250, 250-500 or500-704 amino acids in length).

Embodiments also include HCV peptides that are substantially identicalto those described above. That is, HCV peptides that have one or moreamino acid residues within SEQ. ID. NOs.: 2-11 and fragments thereofthat are substituted by another amino acid of a similar polarity thatacts as a functional equivalent, resulting in a silent alteration.Further, the HCV peptides can have one or more amino acid residues fusedto SEQ. ID. NOs.: 2-11 or a fragment thereof so long as the fusion doesnot significantly alter the structure or function (e.g., immunogenicproperties) of the HCV peptide. Substitutes for an amino acid within thesequence can be selected from other members of the class to which theamino acid belongs. For example, the non-polar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine, and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. The aromatic amino acidsinclude phenylalanine, tryptophan, and tyrosine. Accordingly, thepeptide embodiments of the invention are said to be consistingessentially of SEQ. ID. NOs.: 2-27 in light of the modificationsdescribed above.

The HCV peptides described herein can be prepared by chemical synthesismethods (such as solid phase peptide synthesis) using techniques knownin the art such as those set forth by Merrifield et al., J. Am. Chem.Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA,82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis,Pierce Chem Co., Rockford, Ill. (1984), and Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., all ofwhich are herein expressly incorporated by reference. Such polypeptidescan be synthesized with or without a methionine on the amino terminus.Chemically synthesized HCV peptides can be oxidized using methods setforth in these references to form disulfide bridges.

While the HCV peptides described herein can be chemically synthesized,it can be more effective to produce these polypeptides by recombinantDNA technology. Such methods can be used to construct expression vectorscontaining the HCV nucleotide sequences described above, for example,and appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Alternatively,RNA capable of encoding an HCV nucleotide sequence can be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in Oligonucleotide Synthesis, 1984, Gait, M. J.ed., IRL Press, Oxford, which is incorporated by reference herein in itsentirety. Accordingly, several embodiments concern cell lines that havebeen engineered to express the embodied HCV peptides. For example, somecells are made to express the HCV peptides of SEQ. ID. NOs.: 2-11 orfragments of these molecules (e.g., SEQ. ID. NOs.: 14-26).

A variety of host-expression vector systems can be utilized to expressthe embodied HCV peptides. Suitable expression systems include, but arenot limited to, microorganisms such as bacteria (e.g., E. coli or B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing HCV nucleotide sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the HCV nucleotide sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the HCV sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing HCV sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the HCV geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of HCV peptide or for raising antibodies to the HCVpeptide, for example, vectors which direct the expression of high levelsof fusion protein products that are readily purified can be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., EMBO J., 2:1791 (1983), in which the HCVcoding sequence can be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); VanHeeke & Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the like.pGEX vectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can be purified from lysed cells by adsorptionto glutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The HCV coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of an HCV genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus, (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (See e.g., Smith et al., J. Virol. 46: 584(1983); and Smith, U.S. Pat. No. 4,215,051, herein expresslyincorporated by reference in its entirety).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the HCV nucleotide sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the HCV gene product in infected hosts. (See e.g., Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). Specificinitiation signals can also be required for efficient translation ofinserted HCV nucleotide sequences. These signals include the ATGinitiation codon and adjacent sequences.

However, in cases where only a portion of the HCV coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, can be provided. Furthermore, the initiationcodon can be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (See Bittner et al., Methodsin Enzymol., 153:516-544 (1987)).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products areimportant for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theHCV peptides described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells are allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn arecloned and expanded into cell lines. This method is advantageously usedto engineer cell lines which express the HCV gene product.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223(1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines. (Janknecht, et al., Proc. Natl. Acad. Sci. USA 88: 8972-8976(1991)). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers. The example below describes a method thatwas used to express the HCV peptides encoded by the embodied nucleicacids.

EXAMPLE 8

To characterize the NS3/4A fusion protein, and the truncated and mutatedversions thereof, the vector constructs, described in Example 1, weretranscribed and translated in vitro, and the resulting polypeptides werevisualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). In vitro transcription and translation were performed usingthe T7 coupled reticulocyte lysate system (Promega, Madison, Wis.)according to the manufacturer's instructions. All in vitro translationreactions of the expression constructs were carried out at 30° C. with³⁵S-labeled methionine (Amersham International, Plc, Buckinghamshire,UK). The labeled proteins were separated on 12% SDS-PAGE gels andvisualized by exposure to X-ray film (Hyper Film-MP, Amersham) for 6-18hours.

The in vitro analysis revealed that all proteins were expressed to highamounts from their respective expression constructs. The rNS3 construct(NS3-pVAX vector) produced a single peptide of approximately 61 kDa,whereas, the mutant constructs (e.g., the TGT construct(NS3/4A-TGT-pVAX) and the RGT construct (NS3/4A-RGT-pVAX)) produced asingle polypeptide of approximately 67 kDa, which is identical to themolecular weight of the uncleaved NS3/4A peptide produced from theNS3/4A-pVAX construct. The cleaved product produced from the expressedNS3/4A peptide was approximately 61 kDa, which was identical in size tothe rNS3 produced from the NS3-pVAX vector. These results demonstratedthat the expression constructs were functional, the NS3/4A construct wasenzymatically active, the rNS3 produced a peptide of the predicted size,and the breakpoint mutations completely abolished cleavage at theNS3-NS4A junction.

The sequences, constructs, vectors, clones, and other materialscomprising the embodied HCV nucleic acids and peptides can be inenriched or isolated form. As used herein, “enriched” means that theconcentration of the material is many times its natural concentration,for example, at least about 2, 5, 10, 100, or 1000 times its naturalconcentration, advantageously 0.01%, by weight, preferably at leastabout 0.1% by weight. Enriched preparations from about 0.5% or more, forexample, 1%, 5%, 10%, and 20% by weight are also contemplated. The term“isolated” requires that the material be removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide present ina living animal is not isolated, but the same polynucleotide, separatedfrom some or all of the coexisting materials in the natural system, isisolated. It is also advantageous that the sequences be in purifiedform. The term “purified” does not require absolute purity; rather, itis intended as a relative definition. Isolated proteins have beenconventionally purified to electrophoretic homogeneity by Coomassiestaining, for example. Purification of starting material or naturalmaterial to at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

The HCV gene products described herein can also be expressed in plants,insects, and animals so as to create a transgenic organism. Desirabletransgenic plant systems having an HCV peptide include Arabadopsis,maize, and Chlamydomonas. Desirable insect systems having an HCV peptideinclude, but are not limited to, D. melanogaster and C. elegans. Animalsof any species, including, but not limited to, amphibians, reptiles,birds, mice, hamsters, rats, rabbits, guinea pigs, pigs, micro-pigs,goats, dogs, cats, and non-human primates, e.g., baboons, monkeys, andchimpanzees can be used to generate transgenic animals having anembodied HCV molecule. These transgenic organisms desirably exhibitgermline transfer of HCV peptides described herein.

Any technique known in the art is preferably used to introduce the HCVtransgene into animals to produce the founder lines of transgenicanimals or to knock out or replace existing HCV genes. Such techniquesinclude, but are not limited to pronuclear microinjection (Hoppe, P. C.and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.Sci., USA 82:6148-6152 (1985); gene targeting in embryonic stem cells(Thompson et al., Cell 56:313-321 (1989); electroporation of embryos(Lo, Mol Cell. Biol. 3:1803-1814 (1983); and sperm-mediated genetransfer (Lavitrano et al., Cell 57:717-723 (1989); see also Gordon,Transgenic Animals, Intl. Rev. Cytol. 115:171-229 (1989), all referencesare hereby incorporated by reference herein in their entirety.

Following synthesis or expression and isolation or purification of theHCV peptides, the isolated or purified peptide can be used to generateantibodies. Depending on the context, the term “antibodies” canencompass polyclonal, monoclonal, chimeric, single chain, Fab fragmentsand fragments produced by a Fab expression library. Antibodies thatrecognize the HCV peptides have many uses including, but not limited to,biotechnological applications, therapeutic/prophylactic applications,and diagnostic applications.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, and humans etc. can be immunized by injection withan HCV peptide. Depending on the host species, various adjuvants can beused to increase immunological response. Such adjuvants include, but arenot limited to, ribavirin, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acidsequence consisting of at least four amino acids, and preferably atleast 10 to 15 amino acids. By one approach, short stretches of aminoacids encoding fragments of NS3/4A are fused with those of anotherprotein such as keyhole limpet hemocyanin such that an antibody isproduced against the chimeric molecule. Additionally, a compositioncomprising ribavirin and an HCV peptide (SEQ. ID. NOs.: 2-11), afragment thereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8,10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 4-26), ora nucleic acid encoding one or more of these molecules is administeredto an animal. While antibodies capable of specifically recognizing HCVcan be generated by injecting synthetic 3-mer, 10-mer, and 15-merpeptides that correspond to an HCV peptide into mice, a more diverse setof antibodies can be generated by using recombinant HCV peptides,prepared as decribed above.

To generate antibodies to an HCV peptide, substantially pure peptide isisolated from a transfected or transformed cell. The concentration ofthe peptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibody to the peptide ofinterest can then be prepared as follows:

Monoclonal antibodies to an HCV peptide can be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosboret al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al.Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New YorkN.Y., pp 77-96 (1985). In addition, techniques developed for theproduction of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used. (Morrison etal. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature312:604-608(1984); Takeda et al. Nature 314:452-454(1985).Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceHCV-specific single chain antibodies. Antibodies can also be produced byinducing in vivo production in the lymphocyte population or by screeningrecombinant immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86:3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299(1991).

Antibody fragments that contain specific binding sites for an HCVpeptide can also be generated. For example, such fragments include, butare not limited to, the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).

By one approach, monoclonal antibodies to an HCV peptide are made asfollows. Briefly, a mouse is repetitively inoculated with a fewmicrograms of the selected protein or peptides derived therefrom over aperiod of a few weeks. The mouse is then sacrificed, and the antibodyproducing cells of the spleen isolated. The spleen cells are fused inthe presence of polyethylene glycol with mouse myeloma cells, and theexcess unfused cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare diluted and aliquots of the dilution placed in wells of a microtiterplate where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivativemethods thereof. Selected positive clones can be expanded and theirmonoclonal antibody product harvested for use. Detailed procedures formonoclonal antibody production are described in Davis, L. et al. BasicMethods in Molecular Biology Elsevier, New York. Section 21-2.

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom described above, whichcan be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and can require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections are given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively (e.g., in diagnostic embodimentsthat identify the presence of HCV in biological samples). The nextsection describes how some of the novel nucleic acids and peptidesdescribed above can be used in diagnostics.

Diagnostic Embodiments

Generally, the embodied diagnostics are classified according to whethera nucleic acid or protein-based assay is used. Some diagnostic assaysdetect the presence or absence of an embodied HCV nucleic acid sequencein a sample obtained from a patient, whereas, other assays seek toidentify whether an embodied HCV peptide is present in a biologicalsample obtained from a patient. Additionally, the manufacture of kitsthat incorporate the reagents and methods described herein that allowfor the rapid detection and identification of HCV are also embodied.These diagnostic kits can include, for example, an embodied nucleic acidprobe or antibody, which specifically detects HCV. The detectioncomponent of these kits will typically be supplied in combination withone or more of the following reagents. A support capable of absorbing orotherwise binding DNA, RNA, or protein will often be supplied. Availablesupports include membranes of nitrocellulose, nylon or derivatized nylonthat can be characterized by bearing an array of positively chargedsubstituents. One or more restriction enzymes, control reagents,buffers, amplification enzymes, and non-human polynucleotides likecalf-thymus or salmon-sperm DNA can be supplied in these kits.

Useful nucleic acid-based diagnostics include, but are not limited to,direct DNA sequencing, Southern Blot analysis, dot blot analysis,nucleic acid amplification, and combinations of these approaches. Thestarting point for these analysis is isolated or purified nucleic acidfrom a biological sample obtained from a patient suspected ofcontracting HCV or a patient at risk of contracting HCV. The nucleicacid is extracted from the sample and can be amplified by RT-PCR and/orDNA amplification using primers that correspond to regions flanking theembodied HCV nucleic acid sequences (e.g., NS3/4A (SEQ. ID. NO.: 1)).

In some embodiments, nucleic acid probes that specifically hybridizewith HCV sequences are attached to a support in an ordered array,wherein the nucleic acid probes are attached to distinct regions of thesupport that do not overlap with each other. Preferably, such an orderedarray is designed to be “addressable” where the distinct locations ofthe probe are recorded and can be accessed as part of an assayprocedure. These probes are joined to a support in different knownlocations. The knowledge of the precise location of each nucleic acidprobe makes these “addressable” arrays particularly useful in bindingassays. The nucleic acids from a preparation of several biologicalsamples are then labeled by conventional approaches (e.g., radioactivityor fluorescence) and the labeled samples are applied to the array underconditions that permit hybridization.

If a nucleic acid in the samples hybridizes to a probe on the array,then a signal will be detected at a position on the support thatcorresponds to the location of the hybrid. Since the identity of eachlabeled sample is known and the region of the support on which thelabeled sample was applied is known, an identification of the presenceof the polymorphic variant can be rapidly determined. These approachesare easily automated using technology known to those of skill in the artof high throughput diagnostic or detection analysis.

Additionally, an approach opposite to that presented above can beemployed. Nucleic acids present in biological samples can be disposed ona support so as to create an addressable array. Preferably, the samplesare disposed on the support at known positions that do not overlap. Thepresence of HCV nucleic acids in each sample is determined by applyinglabeled nucleic acid probes that complement nucleic acids, which encodeHCV peptides, at locations on the array that correspond to the positionsat which the biological samples were disposed. Because the identity ofthe biological sample and its position on the array is known, theidentification of a patient that has been infected with HCV can berapidly determined. These approaches are also easily automated usingtechnology known to those of skill in the art of high throughputdiagnostic analysis.

Any addressable array technology known in the art can be employed. Oneparticular embodiment of polynucleotide arrays is known as Genechips™,and has been generally described in U.S. Pat. No. 5,143,854; PCTpublications WO 90/15070 and 92/10092, all of which are herein expresslyincorporated by reference in their entireties. These arrays aregenerally produced using mechanical synthesis methods or light directedsynthesis methods, which incorporate a combination of photolithographicmethods and solid phase oligonucleotide synthesis. (Fodor et al.,Science, 251:767-777, (1991)). The immobilization of arrays ofoligonucleotides on solid supports has been rendered possible by thedevelopment of a technology generally identified as “Very Large ScaleImmobilized Polymer Synthesis” (VLSPIS™) in which, typically, probes areimmobilized in a high density array on a solid surface of a chip.Examples of VLSPIS™ technologies are provided in U.S. Pat. Nos.5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092and WO 95/11995, which describe methods for forming oligonucleotidearrays through techniques such as light-directed synthesis techniques,all of which are herein expressly incorporated by reference in theirentireties. In designing strategies aimed at providing arrays ofnucleotides immobilized on solid supports, further presentationstrategies were developed to order and display the oligonucleotidearrays on the chips in an attempt to maximize hybridization patterns anddiagnostic information. Examples of such presentation strategies aredisclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212, andWO 97/31256, all of which are herein expressly incorporated by referencein their entireties.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid assays. Thereare several ways to produce labeled nucleic acids for hybridization orPCR including, but not limited to, oligolabeling, nick translation,end-labeling, or PCR amplification using a labeled nucleotide.Alternatively, a nucleic acid encoding an HCV peptide can be cloned intoa vector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and can be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3 or SP6 and labeled nucleotides. A number of companies such asPharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and U.S.Biochemical Corp (Cleveland Ohio) supply commercial kits and protocolsfor these procedures. Suitable reporter molecules or labels includethose radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as, substrates, cofactors, inhibitors,magnetic particles and the like.

The presence of an HCV peptide in a protein sample obtained from apatient can also be detected by using conventional assays and theembodiments described herein. For example, antibodies that areimmunoreactive with the disclosed HCV peptides can be used to screenbiological samples for the presence of HCV infection. In preferredembodiments, antibodies that are reactive to the embodied HCV peptidesare used to immunoprecipitate the disclosed HCV peptides from biologicalsamples or are used to react with proteins obtained from a biologicalsample on Western or Immunoblots. Favored diagnostic embodiments alsoinclude enzyme-linked immunosorbant assays (ELISA), radioimmunoassays(RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays(IEMA), including sandwich assays using monoclonal and/or polyclonalantibodies specific for the disclosed HCV peptides. Exemplary sandwichassays are described by David et al., in U.S. Pat. Nos. 4,376,110 and4,486,530, hereby incorporated by reference. Other embodiments employaspects of the immune-strip technology disclosed in U.S. Pat. Nos.5,290,678; 5,604,105; 5,710,008; 5,744,358; and 5,747,274, hereinincorporated by reference.

In another preferred protein-based diagnostic, the antibodies describedherein are attached to a support in an ordered array, wherein aplurality of antibodies are attached to distinct regions of the supportthat do not overlap with each other. As with the nucleic acid-basedarrays, the protein-based arrays are ordered arrays that are designed tobe “addressable” such that the distinct locations are recorded and canbe accessed as part of an assay procedure. These probes are joined to asupport in different known locations. The knowledge of the preciselocation of each probe makes these “addressable” arrays particularlyuseful in binding assays. For example, an addressable array can comprisea support having several regions to which are joined a plurality ofantibody probes that specifically recognize HCV peptides present in abiological sample and differentiate the isotype of HCV identifiedherein.

By one approach, proteins are obtained from biological samples and arethen labeled by conventional approaches (e.g., radioactivity,colorimetrically, or fluorescently). The labeled samples are thenapplied to the array under conditions that permit binding. If a proteinin the sample binds to an antibody probe on the array, then a signalwill be detected at a position on the support that corresponds to thelocation of the antibody-protein complex. Since the identity of eachlabeled sample is known and the region of the support on which thelabeled sample was applied is known, an identification of the presence,concentration, and/or expression level can be rapidly determined. Thatis, by employing labeled standards of a known concentration of HCVpeptide, an investigator can accurately determine the proteinconcentration of the particular peptide in a tested sample and can alsoassess the expression level of the HCV peptide. Conventional methods indensitometry can also be used to more accurately determine theconcentration or expression level of the HCV peptide. These approachesare easily automated using technology known to those of skill in the artof high throughput diagnostic analysis.

In another embodiment, an approach opposite to that presented above canbe employed. Proteins present in biological samples can be disposed on asupport so as to create an addressable array. Preferably, the proteinsamples are disposed on the support at known positions that do notoverlap. The presence of an HCV peptide in each sample is thendetermined by applying labeled antibody probes that recognize epitopesspecific for the HCV peptide. Because the identity of the biologicalsample and its position on the array is known, an identification of thepresence, concentration, and/or expression level of an HCV peptide canbe rapidly determined.

That is, by employing labeled standards of a known concentration of HCVpeptide, an investigator can accurately determine the concentration ofpeptide in a sample and from this information can assess the expressionlevel of the peptide. Conventional methods in densitometry can also beused to more accurately determine the concentration or expression levelof the HCV peptide. These approaches are also easily automated usingtechnology known to those of skill in the art of high throughputdiagnostic analysis. As detailed above, any addressable array technologyknown in the art can be employed. The next section describes morecompositions that involve the embodied HCV nucleic acids and/or HCVpeptides.

Compositions Comprising HCV Nucleic Acids or Peptides

Embodiments of the invention also include NS3/4A fusion proteins ornucleic acids encoding these molecules. For instance, production andpurification of recombinant protein may be facilitated by the additionof auxiliary amino acids to form a “tag”. Such tags include, but are notlimited to, His-6, Flag, Myc and GST. The tags may be added to theC-terminus, N-terminus, or within the NS3/4A amino acid sequence.Further embodiments include NS3/4A fusion proteins with amino or carboxyterminal truncations, or internal deletions, or with additionalpolypeptide sequences added to the amino or carboxy terminal ends, oradded internally. Other embodiments include NS3/4A fusion proteins, ortruncated or mutated versions thereof, where the residues of the NS3/4Aproteolytic cleavage site have been substituted. Such substitutionsinclude, but are not limited to, sequences where the P1′ site is a Ser,Gly, or Pro, or the P1 position is an Arg, or where the P8 to P4′sequence is Ser-Ala-Asp-Leu-Glu-Val-Val-Thr-Ser-Thr-Trp-Val (SEQ. ID.NO.: 15).

More embodiments concern an immunogen comprising the NS3/4A fusionprotein, or a truncated, mutated, or modified version thereof, capableof eliciting an enhanced immune response against NS3. The immunogen canbe provided in a substantially purified form, which means that theimmunogen has been rendered substantially free of other proteins,lipids, carbohydrates or other compounds with which it naturallyassociates.

Some embodiments contain at least one of the HCV nucleic acids or HCVpeptides (e.g., SEQ. ID. NOs.: 1-27) joined to a support. Preferably,these supports are manufactured so as to create a multimeric agent.These multimeric agents provide the HCV peptide or nucleic acid in sucha form or in such a way that a sufficient affinity to the molecule isachieved. A multimeric agent having an HCV nucleic acid or peptide canbe obtained by joining the desired molecule to a macromolecular support.A “support” can be a termed a carrier, a protein, a resin, a cellmembrane, or any macromolecular structure used to join or immobilizesuch molecules. Solid supports include, but are not limited to, thewalls of wells of a reaction tray, test tubes, polystyrene beads,magnetic beads, nitrocellulose strips, membranes, microparticles such aslatex particles, animal cells, Duracyte®, artificial cells, and others.An HCV nucleic acid or peptide can also be joined to inorganic carriers,such as silicon oxide material (e.g., silica gel, zeolite, diatomaceousearth or aminated glass) by, for example, a covalent linkage through ahydroxy, carboxy or amino group and a reactive group on the carrier.

In several multimeric agents, the macromolecular support has ahydrophobic surface that interacts with a portion of the HCV nucleicacid or peptide by a hydrophobic non-covalent interaction. In somecases, the hydrophobic surface of the support is a polymer such asplastic or any other polymer in which hydrophobic groups have beenlinked such as polystyrene, polyethylene or polyvinyl. Additionally, HCVnucleic acid or peptide can be covalently bound to carriers includingproteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen,chitosane or aminated sepharose). In these later multimeric agents, areactive group on the molecule, such as a hydroxy or an amino group, isused to join to a reactive group on the carrier so as to create thecovalent bond. Additional multimeric agents comprise a support that hasother reactive groups that are chemically activated so as to attach theHCV nucleic acid or peptide. For example, cyanogen bromide activatedmatrices, epoxy activated matrices, thio and thiopropyl gels,nitrophenyl chloroformate and N-hydroxy succinimide chlorformatelinkages, or oxirane acrylic supports are used. (Sigma).

Carriers for use in the body, (i.e. for prophylactic or therapeuticapplications) are desirably physiological, non-toxic and preferably,non-immunoresponsive. Suitable carriers for use in the body includepoly-L-lysine, poly-D, L-alanine, liposomes, and Chromosorb®(Johns-Manville Products, Denver Colo.). Ligand conjugated Chromosorb®(Synsorb-Pk) has been tested in humans for the prevention ofhemolytic-uremic syndrome and was reported as not presenting adversereactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045(1995)). For some embodiments, a “naked” carrier (i.e., lacking anattached HCV nucleic acid or peptide) that has the capacity to attach anHCV nucleic acid or peptide in the body of a organism is administered.By this approach, a “prodrug-type” therapy is envisioned in which thenaked carrier is administered separately from the HCV nucleic acid orpeptide and, once both are in the body of the organism, the carrier andthe HCV nucleic acid or peptide are assembled into a multimeric complex.

The insertion of linkers, such as linkers (e.g., “λ linkers” engineeredto resemble the flexible regions of λ phage) of an appropriate lengthbetween the HCV nucleic acid or peptide and the support are alsocontemplated so as to encourage greater flexibility of the HCV peptide,hybrid, or binding partner and thereby overcome any steric hindrancethat can be presented by the support. The determination of anappropriate length of linker that allows for an optimal cellularresponse or lack thereof, can be determined by screening the HCV nucleicacid or peptide with varying linkers in the assays detailed in thepresent disclosure.

A composite support comprising more than one type of HCV nucleic acid orpeptide is also envisioned. A “composite support” can be a carrier, aresin, or any macromolecular structure used to attach or immobilize twoor more different HCV nucleic acids or peptides. As above, the insertionof linkers, such as λ linkers, of an appropriate length between the HCVnucleic acid or peptide and the support is also contemplated so as toencourage greater flexibility in the molecule and thereby overcome anysteric hindrance that can occur. The determination of an appropriatelength of linker that allows for an optimal cellular response or lackthereof, can be determined by screening the HCV nucleic acid or peptidewith varying linkers in the assays detailed in the present disclosure.

In other embodiments, the multimeric and composite supports discussedabove can have attached multimerized HCV nucleic acids or peptides so asto create a “multimerized-multimeric support” and a“multimerized-composite support”, respectively. A multimerized ligandcan, for example, be obtained by coupling two or more HCV nucleic acidsor peptides in tandem using conventional techniques in molecularbiology. The multimerized form of the HCV nucleic acid or peptide can beadvantageous for many applications because of the ability to obtain anagent with a higher affinity, for example. The incorporation of linkersor spacers, such as flexible λ linkers, between the individual domainsthat make-up the multimerized agent can also be advantageous for someembodiments. The insertion of λ linkers of an appropriate length betweenprotein binding domains, for example, can encourage greater flexibilityin the molecule and can overcome steric hindrance. Similarly, theinsertion of linkers between the multimerized HCV nucleic acid orpeptide and the support can encourage greater flexibility and limitsteric hindrance presented by the support. The determination of anappropriate length of linker can be determined by screening the HCVnucleic acids or peptides in the assays detailed in this disclosure.

Embodiments also include vaccine compositions comprising the NS3/4Afusion protein, or a truncated or mutated version thereof, and,optionally, an adjuvant. The next section describes some of thepreferred vaccine compositions in greater detail.

Vaccine Compositions

Vaccine compositions comprising either an embodied HCV nucleic acid orHCV peptide or both (e.g., any one or more of SEQ. ID. NOs.: 1-27 arecontemplated. These compositions typically contain an adjuvant, but donot necessarily require an adjuvant. That is many of the nucleic acidsand peptides described herein function as immunogens when administeredneat. The compositions described herein (e.g., the HCV immunogens andvaccine compositions containing an adjuvant, such as ribavirin) can bemanufactured in accordance with conventional methods of galenic pharmacyto produce medicinal agents for administration to animals, e.g., mammalsincluding humans.

Various nucleic acid-based vaccines are known and it is contemplatedthat these compositions and approaches to immunotherapy can be augmentedby reformulation with ribavirin (See e.g., U.S. Pat. Nos. 5,589,466 and6,235,888, both of which are herein expressly incorporated by referencein their entireties). By one approach, for example, a gene encoding oneof the HCV peptides described herein (e.g., SEQ. ID. NO.: 1) is clonedinto an expression vector capable of expressing the polypeptide whenintroduced into a subject. The expression construct is introduced intothe subject in a mixture of adjuvant (e.g., ribavirin) or in conjunctionwith an adjuvant (e.g., ribavirin). For example, the adjuvant (e.g.,ribavirin) is administered shortly after the expression construct at thesame site. Alternatively, RNA encoding the HCV polypeptide antigen ofinterest is provided to the subject in a mixture with ribavirin or inconjunction with an adjuvant (e.g., ribavirin).

Where the antigen is to be DNA (e.g., preparation of a DNA vaccinecomposition), suitable promoters include Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human actin, human myosin, human hemoglobin, humanmuscle creatine and human metalothionein can be used. Examples ofpolyadenylation signals useful with some embodiments, especially in theproduction of a genetic vaccine for humans, include but are not limitedto, SV40 polyadenylation signals and LTR polyadenylation signals. Inparticular, the SV40 polyadenylation signal, which is in pCEP4 plasmid(Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylationsignal, is used.

In addition to the regulatory elements required for gene expression,other elements may also be included in a gene construct. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human actin, human myosin, humanhemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV. Gene constructs can be provided with mammalian originof replication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Plasmids pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region, whichproduces high copy episomal replication without integration. All formsof DNA, whether replicating or non-replicating, which do not becomeintegrated into the genome, and which are expressible, can be used.Preferably, the genetic vaccines comprise ribavirin and a nucleic acidencoding NS3/4A, NS3, or a fragment or mutant thereof (SEQ. ID. NOs.:2-26). The following example describes the preparation of a geneticvaccine suitable for use in humans.

EXAMPLE 9

An HCV expression plasmid is designed to express the NS3/4A peptide(SEQ. ID. NO.: 2). The NS3/4A coding sequence of NS3/4A-pVAX is removedby digestion with EcoRI and XbaI, and the isolated fragment is insertedinto plasmid A so that it is under the transcriptional control of theCMV promoter and the RSV enhancer element. (See U.S. Pat. No. 6,235,888to Pachuk, et al., herein expressly incorporated by reference in itsentirety). Plasmid Plasmid backbone A is 3969 base pairs in length; itcontains a PBR origin of replication for replicating in E. coli and akanamycin resistance gene. Inserts such as the NS3/4A, are cloned into apolylinker region, which places the insert between and operably linkedto the promoter and polyadenylation signal. Transcription of the clonedinserts is under the control of the CMV promoter and the RSV enhancerelements. A polyadenylation signal is provided by the presence of anSV40 poly A signal situated just 3′ of the cloning site. An NS3/4Acontaining vaccine composition is then made by mixing 500 μg of therNS3/4A construct with 1 mg of ribavirin.

Said vaccine composition can be used to raise antibodies in a mammal(e.g., mice or rabbits) or can be injected intramuscularly into a humanso as to to raise antibodies, preferably a human that is chronicallyinfected with the HCV virus. The recipient preferably receives threeimmunization boosts of the mixture at 4-week intervals, as well. By thethird boost, the titer of antibody specific for HCV will besignificantly increased. Additionally, at this time, said subject willexperience an enhanced antibody and T-cell mediated immune responseagainst NS3, as evidenced by an increased fraction of NS3 specificantibodies as detected by EIA, and a reduction in viral load as detectedby RT-PCR.

Also contemplated are vaccine compositions comprising one or more of theHCV peptides described herein. Preferably, the embodied peptide vaccinescomprise ribavirin and NS3/4A, NS3, or a fragment or mutant thereof(e.g., SEQ. ID. NOs.: 2-26). The following example describes an approachto prepare a vaccine composition comprising an NS3/4A fusion protein andan adjuvant.

EXAMPLE 10

To generate a tagged NS3/4A construct, the NS3/4A coding sequence ofNS3/4A-pVAX is removed by digestion with EcoRI and XbaI, and theisolated fragment is inserted into an Xpress vector (Invitrogen). TheXpress vector allows for the production of a recombinant fusion proteinhaving a short N-terminal leader peptide that has a high affinity fordivalent cations. Using a nickel-chelating resin (Invitrogen), therecombinant protein can be purified in one step and the leader can besubsequently removed by cleavage with enterokinase. A preferred vectoris the pBlueBacHis2 Xpress. The pBlueBacHis2 Xpress vector is aBaculovirus expression vector containing a multiple cloning site, anampicillin resistance gene, and a lac z gene. Accordingly, the digestedamplification fragment is cloned into the pBlueBacHis2 Xpress vector andSF9 cells are infected. The expression protein is then isolated orpurified according to the manufacturer's instructions. An NS3/4Acontaining vaccine composition is then made by mixing 100 μg of therNS3/4A with 1 mg of ribavirin.

Said vaccine composition can be used to raise antibodies in a mammal(e.g., mice or rabbits) or can be injected intramuscularly into a humanso as to to raise antibodies, preferably a human that is chronicallyinfected with the HCV virus. The recipient preferably receives threeimmunization boosts of the mixture at 4-week intervals. By the thirdboost, the titer of antibody specific for HCV will be significantlyincreased. Additionally, at this time, said subject will experience anenhanced antibody and T-cell mediated immune response against NS3, asevidenced by an increased fraction of NS3 specific antibodies asdetected by EIA, and a reduction in viral load as detected by RT-PCR.

The compositions (e.g., vaccines) that comprise one or more of theembodied HCV nucleic acids or peptides may contain other ingredientsincluding, but not limited to, adjuvants (e.g., ribavirin), bindingagents, excipients such as stabilizers (to promote long term storage),emulsifiers, thickening agents, salts, preservatives, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. These compositionsare suitable for treatment of animals either as a preventive measure toavoid a disease or condition or as a therapeutic to treat animalsalready afflicted with a disease or condition.

Many other ingredients can be also be present. For example, the adjuvant(e.g., ribavirin) and antigen can be employed in admixture withconventional excipients (e.g., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with theribavirin and/or antigen). Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols, gumarabic, vegetable oils, benzyl alcohols, polyetylene glycols, gelatine,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Many more suitablecarriers are described in Remmington's Pharmaceutical Sciences, 15thEdition, Easton:Mack Publishing Company, pages 1405-1412 and1461-1487(1975) and The National Formulary XIV, 14th Edition,Washington, American Pharmaceutical Association (1975), herein expresslyincorporated by reference in their entireties.

The gene constructs described herein, in particular, may be formulatedwith or administered in conjunction with agents that increase uptakeand/or expression of the gene construct by the cells relative to uptakeand/or expression of the gene construct by the cells that occurs whenthe identical genetic vaccine is administered in the absence of suchagents. Such agents and the protocols for administering them inconjunction with gene constructs are described in PCT Patent ApplicationSerial Number PCT/US94/00899 filed Jan. 26, 1994, which is incorporatedherein by reference. Examples of such agents include: CaPO₄, DEAEdextran, anionic lipids; extracellular matrix-active enzymes; saponins;lectins; estrogenic compounds and steroidal hormones; hydroxylated loweralkyls; dimethyl sulfoxide (DMSO); urea; and benzoic acid estersanilides, amidines, urethanes and the hydrochloride salts thereof suchas those of the family of local anesthetics. In addition, the geneconstructs are encapsulated within/administered in conjunction withlipids/polycationic complexes.

Vaccines can be sterilized and if desired mixed with auxiliary agents,e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like that do notdeleteriously react with the adjuvant (e.g., ribavirin) or the HCVnucleic acid or peptide.

The effective dose and method of administration of a particular vaccineformulation can vary based on the individual patient and the type andstage of the disease, as well as other factors known to those of skillin the art. Therapeutic efficacy and toxicity of the vaccines can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population). The data obtained from cell culture assays andanimal studies can be used to formulate a range of dosage for human use.The dosage of the vaccines lies preferably within a range of circulatingconcentrations that include the ED₅₀ with no toxicity. The dosage varieswithin this range depending upon the type of adjuvant derivative and HCVantigen, the dosage form employed, the sensitivity of the patient, andthe route of administration.

Since many adjuvants, including ribavirin, has been on the market forseveral years, many dosage forms and routes of administration are known.All known dosage forms and routes of administration can be providedwithin the context of the embodiments described herein. Preferably, anamount of adjuvant (e.g., ribavirin) that is effective to enhance animmune response to an antigen in an animal can be considered to be anamount that is sufficient to achieve a blood serum level of antigenapproximately 0.25-12.5 μg/ml in the animal, preferably, about 2.5μg/ml. In some embodiments, the amount of adjuvant (e.g., ribavirin) isdetermined according to the body weight of the animal to be given thevaccine. Accordingly, the amount of adjuvant (e.g., ribavirin) in avaccine formulation can be from about 0.1-6.0 mg/kg body weight. Thatis, some embodiments have an amount of adjuvant (e.g., ribavirin) thatcorresponds to approximately 0.1-1.0 mg/kg, 1.1-2.0 mg/kg, 2.1-3.0mg/kg, 3.1-4.0 mg/kg, 4.1-5.0 mg/kg, 5.1, and 6.0 mg/kg body weight ofan animal. More conventionally, the vaccines contain approximately 0.25mg -2000 mg of adjuvant (e.g., ribavirin). That is, some embodimentshave approximately 250 μg, 500 μg, 1 mg, 25 mg, 50 mg, 100 mg, 150 mg,200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg,650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, and 2 g of adjuvant (e.g.,ribavirin).

As one of skill in the art will appreciate, the amount of antigens in avaccine can vary depending on the type of antigen and itsimmunogenicity. The amount of antigens in the vaccines can varyaccordingly. Nevertheless, as a general guide, the vaccines can haveapproximately 0.25 mg-5 mg, 5-10 mg, 10-100 mg, 100-500 mg, and upwardsof 2000 mg of an HCV antigen described herein. Preferably, the amount ofHCV antigen is 0.1 μg -1 mg, desirably, 1 μg-100 g, preferably 5 μg-50μg, and, most preferably, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg-20 μg, whensaid antigen is a nucleic acid and 1 μg-100 mg, desirably, 10 μg-10 mg,preferably, 100 μg-1 mg, and, most preferably, 200 μg, 300 μg, 400 μg,500 μg, 600 μg, or 700 μg-1 mg, when said antigen is a peptide.

In some approaches described herein, the exact amount of adjuvant (e.g.,ribavirin) and/or HCV antigen is chosen by the individual physician inview of the patient to be treated. Further, the amounts of ribavirin canbe added in combination to or separately from the same or equivalentamount of antigen and these amounts can be adjusted during a particularvaccination protocol so as to provide sufficient levels in light ofpatient-specific or antigen-specific considerations. In this vein,patient-specific and antigen-specific factors that can be taken intoaccount include, but are not limited to, the severity of the diseasestate of the patient, age, and weight of the patient, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. The next sectiondescribes the discovery that ribavirin is an effective adjuvant.

Ribavirin

Nucleoside analogs have been widely used in anti-viral therapies due totheir capacity to reduce viral replication. (Hosoya et al., J. Inf.Dis., 168:641-646 (1993)). ribavirin(1-β-D-riboftiranosyl-1,2,4-triazole-3-carboxamide) is a syntheticguanosine analog that has been used to inhibit RNA and DNA virusreplication. (Huffman et al., Antimicrob. Agents. Chemother., 3:235(1973); Sidwell et al., Science, 177:705 (1972)). Ribavirin has beenshown to be a competitive inhibitor of inositol mono-phosphate (IMP)dehydrogenase (IMPDH), which converts IMP to IMX (which is thenconverted to GMP). De Clercq, Anti viral Agents: characteristic activityspectrum depending on the molecular target with which they interact,Academic press, Inc., New York N.Y., pp. 1-55 (1993). Intracellularpools of GTP become depleted as a result of long term ribavirintreatment.

In addition to antiviral activity, investigators have observed that someguanosine analogs have an effect on the immune system. (U.S. Pat. Nos.6,063,772 and 4,950,647). Ribavirin has been shown to inhibit functionalhumoral immune responses (Peavy et al., J. Immunol., 126:861-864 (1981);Powers et al., Antimicrob. Agents. Chemother., 22:108-114 (1982)) andIgE-mediated modulation of mast cell secretion. (Marquardt et al., J.Pharmacol. Exp. Therapeutics, 240:145-149 (1987)). Some investigatorsreport that a daily oral therapy of ribavirin has an immune modulatingeffect on humans and mice. (Hultgren et al., J. Gen. Virol.,79:2381-2391 (1998) and Cramp et al., Gastron. Enterol., 118:346-355(2000)). Nevertheless, the current understanding of the effects ofribavirin on the immune system is in its infancy. As disclosed inExamples 9-12, ribavirin was found to be a potent adjuvant.

EXAMPLE 11

In a first set of experiments, groups of three to five Balb/c mice (BKUniversal, Uppsala, Sweden) were immunized i.p or s.c. (e.g., at thebase of the tail) with 10 μg or 100 μg of recombinant hepatitis C virusnon-structural 3 (rNS3) protein. The rNS3 was dissolved in phosphatebuffered saline (PBS) alone or PBS containing 1 mg ribavirin (obtainedfrom ICN, Costa Mesa, Calif.). Mice were injected with a total volume of100 μl per injection.

At two and four weeks following i.p. immunization, all mice were bled byretro-orbital sampling. Serum samples were collected and analyzed forthe presence of antibodies to rNS3. To determine the antibody titer, anenzyme immunoassay (EIA) was performed. (See e.g., Hultgren et al., JGen Virol. 79:2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab.Immunol. 4:630-632 (1997), both of which are herein expresslyincorporated by reference in their entireties). The antibody levels wererecorded as the highest serum dilution giving an optical density at 405nm more than twice that of non-immunized mice.

Mice that received 10 μg or 100 μg rNS3 mixed with 1 mg ribavirin in PBSdisplayed consistently higher levels of NS3 antibodies. The antibodytiter that was detected by EIA at two weeks post-immunization is shownin FIG. 3. The vaccine formulations having 1 mg of ribavirin and either10 μg or 100 μg of rNS3 induced a significantly greater antibody titerthan the vaccine formulations composed of only rNS3.

In a second set of experiments, groups of eight Balb/c mice wereimmunized intraperitoneally with 10 or 50 μg of rNS3 in 100 μl phosphatebuffered saline containing either 0 mg, 1 mg, 3 mg, or 10 mg ribavirin(Sigma). At four, six and eight weeks the mice were bled and serum wasseparated and frozen. After completion of the study, sera were testedfor the levels of antibodies to recombinant NS3, as described above.Mean antibody levels to rNS3 were compared between the groups usingStudent's t-test (parametric analysis) or Mann-Whitney (non-parametricanalysis) and the software package StatView 4.5 (Abacus Concepts,Berkely, Calif.). The adjuvant effect of ribavirin when added in threedoses to 10 μg of rNS3 are provided in TABLE 7. The adjuvant effect ofribavirin when added in three doses to 50 μg of rNS3 are provided inTABLE 7. Parametrical comparison of the mean rNS3 antibody titres inmice receiving different 10 μg or 50 μg of rNS3 and different doses ofribavirin are provided in TABLES 8 and 9, respectively. Non-parametricalcomparison of mean NS3 antibody titres in mice receiving different 10 μgor 50 μg of rNS3 and different doses of ribavirin are provided in TABLES10 and 11, respectively. The values given represent end point titres torecombinant rNS3.

TABLE 7 Amount Amount Antibody titre to rNS3 ribavirin immunogen Mouseat indicated week (mg/dose) (μg/dose) ID Week 4 Week 6 Week 8 None 105:1 300 1500 1500 None 10 5:2 <60 7500 1500 None 10 5:3 <60 1500 300None 10 5:4 60 1500 1500 None 10 5:5 <60 1500 nt None 10 5:6 60 15001500 None 10 5:7 <60 7500 7500 None 10 5:8 300 37500 7500 Group meantitre (mean ± SD) 180 ± 139  7500 ± 12421 3042 ± 3076 1 10 6:1 300 3750037500 1 10 6:2 <60 1500 1500 1 10 6:3 300 37500 187500 1 10 6:4 30037500 7500 1 10 6:5 60 nt nt 1 10 6:6 <60 37500 7500 1 10 6:7 <60 375007500 1 10 6:8 300 7500 7500 Group mean titre (mean ± SD) 252 ± 107 28071± 16195 36642 ± 67565 3 10 7:1 60 37500 7500 3 10 7:2 60 37500 37500 310 7:3 300 7500 7500 3 10 7:4 300 37500 7500 3 10 7:5 300 37500 37500 310 7:6 300 37500 37500 3 10 7:7 60 7500 7500 3 10 7:8 60 37500 37500Group mean titre (mean ± SD) 180 ± 128 30000 ± 13887 22500 ± 34637 10 108:1 300 37500 37500 10 10 8:2 300 37500 37500 10 10 8:3 <60 300 300 1010 8:4 60 7500 7500 10 10 8:5 <60 300 300 10 10 8:6 <60 37500 37500 1010 8:7 <60 7500 7500 10 10 8:8 <60 nt nt Group mean titre (mean ± SD)220 ± 139 18300 ± 18199 18300 ± 18199

TABLE 8 Amount Amount Antibody titre to rNS3 ribavirin immunogen Mouseat indicated week (mg/dose) (μg/dose) ID Week 4 Week 6 Week 8 None 501:1 60 7500 7500 None 50 1:2 60 7500 7500 None 50 1:3 60 7500 7500 None50 1:4 <60 1500 300 None 50 1:5 300 37500 37500 None 50 1:6 60 7500 7500None 50 1:7 60 37500 7500 None 50 1:8 — — — Group mean titre (mean ± SD)100 ± 98  15214 ± 15380 10757 ± 12094 1 50 2:1 60 7500 7500 1 50 2:2 30037500 7500 1 50 2:3 60 187500 7500 1 50 2:4 60 37500 187500 1 50 2:5 6037500 7500 1 50 2:6 60 37500 37500 1 50 2:7 300 37500 7500 1 50 2:8 30037500 37500 Group mean titre (mean ± SD) 150 ± 124 52500 ± 55549 37500 ±62105 3 50 3:1 60 37500 7500 3 50 3:2 300 37500 37500 3 50 3:3 300 375007500 3 50 3:4 60 37500 7500 3 50 3:5 300 37500 7500 3 50 3:6 60 375007500 3 50 3:7 — 7500 37500 3 50 3:8 1500 7500 37500 Group mean titre(mean ± SD) 387 ± 513 30000 ± 13887 18750 ± 15526 10 50 4:1 300 75007500 10 50 4:2 300 37500 37500 10 50 4:3 60 7500 7500 10 50 4:4 60 75007500 10 50 4:5 60 1500 1500 10 50 4:6 60 7500 37500 10 50 4:7 — 75007500 10 50 8:8 60 37500 7500 Group mean titre (mean ± SD) 140 ± 12410929 ± 11928 15214 ± 15380

TABLE 9 Group Week Mean ± SD Group Mean ± SD analysis p-value 10 μgNS3/no 4 180 ± 139 10 μg NS3/ 252 ± 107 Students t-test 0.4071 ribavirin 1 mg ribavirin 6  7500 ± 12421 28071 ± 16195 Students t-test 0.0156* 83042 ± 3076 36642 ± 67565 Students t-test 0.2133 10 μg NS3/no 4 180 ±139 10 μg NS3/ 180 ± 128 Students t-test 1.000 ribavirin  3 mg ribavirin6  7500 ± 12421 30000 ± 13887 Students t-test 0.0042** 8 3042 ± 307622500 ± 34637 Students t-test 0.0077** 10 μg NS3/no 4 180 ± 139 10 μgNS3/ 220 ± 139 Students t-test 0.7210 ribavirin 10 mg ribavirin 6  7500± 12421 18300 ± 18199 Students t-test 0.1974 8 3042 ± 3076 18300 ± 18199Students t-test 0.0493*

TABLE 10 Group Week Mean ± SD Group Mean ± SD analysis p-value 50 μgNS3/no 4 100 ± 98  50 μg NS3/ 150 ± 124 Students t-test 0.4326 ribavirin 1 mg ribavirin 6 15214 ± 15380 52500 ± 55549 Students t-test 0.1106 810757 ± 12094 37500 ± 62105 Students t-test 0.2847 50 μg NS3/no 4 100 ±98  50 μg NS3/ 387 ± 513 Students t-test 0.2355 ribavirin  3 mgribavirin 6 15214 ± 15380 30000 ± 13887 Students t-test 0.0721 8 10757 ±12094 18750 ± 15526 Students t-test 0.2915 50 μg NS3/no 4 100 ± 98  50μg NS3/ 140 ± 124 Students t-test 0.5490 ribavirin 10 mg ribavirin 615214 ± 15380 10929 ± 11928 Students t-test 0.5710 8 10757 ± 12094 15214± 15380 Students t-test 0.5579 Significance levels: NS = notsignificant; *= p < 0.05; **= p < 0.01; ***= p < 0.001

TABLE 11 Group Week Mean ± SD Group Mean ± SD analysis p-value 10 μgNS3/no 4 180 ± 139 10 μg NS3/ 252 ± 107 Mann-Whitney 0.4280 ribavirin  1mg ribavirin 6  7500 ± 12421 28071 ± 16195 Mann-Whitney 0.0253* 8 3042 ±3076 36642 ± 67565 Mann-Whitney 0.0245* 10 μg NS3/no 4 180 ± 139 10 μgNS3/ 180 ± 128 Mann-Whitney 0.0736 ribavirin  3 mg ribavirin 6  7500 ±12421 30000 ± 13887 Mann-Whitney 0.0050** 8 3042 ± 3076 22500 ± 34637Mann-Whitney 0.0034** 10 μg NS3/no 4 180 ± 139 10 μg NS3/ 220 ± 139Mann-Whitney 0.8986 ribavirin 10 mg ribavirin 6  7500 ± 12421 18300 ±18199 Mann-Whitney 0.4346 8 3042 ± 3076 18300 ± 18199 Mann-Whitney0.2102

TABLE 12 Group Week Mean ± SD Group Mean ± SD analysis p-value 50 μgNS3/no 4 100 ± 98  50 μg NS3/ 150 ± 124 Mann-Whitney 0.1128 ribavirin  1mg ribavirin 6 15214 ± 15380 52500 ± 55549 Mann-Whitney 0.0210* 8 10757± 12094 37500 ± 62105 Mann-Whitney 0.1883 50 μg NS3/no 4 100 ± 98  50 μgNS3/ 387 ± 513 Mann-Whitney 0.1400 ribavirin  3 mg ribavirin 6 15214 ±15380 30000 ± 13887 Mann-hitney 0.0679 8 10757 ± 12094 18750 ± 15526Mann-Whitney 0.2091 50 μg NS3/no 4 100 ± 98  50 μg NS3/ 140 ± 124Mann-Whitney 0.4292 ribavirin 10 mg ribavirin 6 15214 ± 15380 10929 ±11928 Mann-Whitney 0.9473 8 10757 ± 12094 15214 ± 15380 Mann-Whitney0.6279 Significance levels: NS = not significant; *= p < 0.05; **= p <0.01; ***= p < 0.001

The data above demonstrates that ribavirin facilitates or enhances animmune response to an HCV antigen or HCV epitopes. A potent immuneresponse to rNS3 was elicited after immunization with a vaccinecomposition comprising as little as 1 mg ribavirin and 10 μg of rNS3antigen. The data above also provide evidence that the amount ofribavirin that is sufficient to facilitate an immune response to anantigen is between 1 and 3 mg per injection for a 25-30 g Balb/c mouse.It should be realized, however, that these amounts are intended forguidance only and should not be interpreted to limit the scope of theinvention in any way. Nevertheless, the data shows that vaccinecompositions comprising approximately 1 to 3 mg doses of ribavirininduce an immune response that is more than 12 times higher than theimmune response elicited in the absence of without ribavirin. Thus,ribavirin has a significant adjuvant effect on the humoral immuneresponse of an animal and thereby, enhances or facilitates the immuneresponse to the antigen. The example below describes experiments thatwere performed to better understand the amount of ribavirin needed toenhance or facilitate an immune response to an antigen.

EXAMPLE 12

To determine a dose of ribavirin that is sufficient to provide anadjuvant effect, the following experiments were performed. In a firstset of experiments, groups of mice (three per group) were immunized witha 20 μg rNS3 alone or a mixture of 20 μg rNS3 and 0.1 mg, 1 mg, or 10 mgribavirin. The levels of antibody to the antigen were then determined byEIA. The mean endpoint titers at weeks 1 and 3 were plotted and areshown in FIG. 4. It was discovered that the adjuvant effect provided byribavirin had different kinetics depending on the dose of ribavirinprovided. For example, even low doses (<1 mg) of ribavirin were found toenhance antibody levels at week one but not at week three, whereas,higher doses (1-10 mg) were found to enhance antibody levels at weekthree.

A second set of experiments was also performed. In these experiments,groups of mice were injected with vaccine compositions comprisingvarious amounts of ribavirin and rNS3 and the IgG response in theseanimals was monitored. The vaccine compositions comprised approximately100 μl phosphate buffered saline and 20 μg rNS3 with or without 0.1 mg,1.0 mg, or 10 mg ribavirin (Sigma). The mice were bled at week six andrNS3-specific IgG levels were determined by EIA as described previously.As shown in TABLE 13, the adjuvant effects on the sustained antibodylevels were most obvious in the dose range of 1 to 10 mg per injectionfor a 25-30 g mouse.

TABLE 13 Amount (mg) ribavirin Endpoint titre of rNS3 IgG at indicatedweek Immunogen mixed with the immunogen Mouse ID Week 1 Week 2 Week 3 20μg rNS3 None 1 60 360 360 20 μg rNS3 None 2 360 360 2160 20 μg rNS3 None3 360 2160 2160 Mean 260 ± 173  960 ± 1039 1560 ± 1039 20 μg rNS3 0.1 42160 12960 2160 20 μg rNS3 0.1 5 60 60 60 20 μg rNS3 0.1 6 <60 2160 21601110 ± 1484 5060 ± 6921 1460 ± 1212 20 μg rNS3 1.0 7 <60 60 12960 20 μgrNS3 1.0 8 <60 2160 2160 20 μg rNS3 1.0 9 360 2160 2160 Mean 360 1460 ±1212 5760 ± 6235 20 μg rNS3 10.0 10 360 12960 77760 20 μg rNS3 10.0 11<60 2160 12960 20 μg rNS3 10.0 12 360 2160 2160 Mean 360 5760 ± 623530960 ± 40888

In a third set of experiments, the adjuvant effect of ribavirin afterprimary and booster injections was investigated. In these experiments,mice were given two intraperitoneal injections of a vaccine compositioncomprising 10 μg rNS3 with or without ribavirin and the IgG subclassresponses to the antigen was monitored, as before. Accordingly, micewere immunized with 100 μl phosphate buffered containing 10 μgrecombinant NS3 alone, with or without 0.1 or 1.0 mg ribavirin (Sigma)at weeks 0 and 4. The mice were bled at week six and NS3-specific IgGsubclasses were determined by EIA as described previously. As shown inTABLE 14, the addition of ribavirin to the immunogen prior to theinjection does not change the IgG subclass response in the NS3-specificimmune response. Thus, the adjuvant effect of a vaccine compositioncomprising ribavirin and an antigen can not be explained by a shift inof the Th1/Th2-balance. It appears that another mechanism may beresponsible for the adjuvant effect of ribavirin.

TABLE 14 Amount (mg) ribavirin Endpoint titre of indicated NS3 IgGsubclass Immunogen mixed with the immunogen Mouse ID IgG1 IgG2a IgG2bIgG3 10 μg rNS3 None 1 360 60 <60 60 10 μg rNS3 None 2 360 <60 <60 60 10μg rNS3 None 3 2160 60 <60 360 Mean 960 ± 1039 60 — 160 ± 173 10 μg rNS30.1 4 360 <60 <60 60 10 μg rNS3 0.1 5 60 <60 <60 <60 10 μg rNS3 0.1 62160 60 60 360 860 ± 1136 60 60 210 ± 212 10 μg rNS3 1.0 7 2160 <60 <6060 10 μg rNS3 1.0 8 360 <60 <60 <60 10 μg rNS3 1.0 9 2160 <60 <60 60Mean 1560 ± 1039  — — 60

The data presented in this example further verify that ribavirin can beadministered as an adjuvant and establish that that the dose ofribavirin can modulate the kinetics of the adjuvant effect. The examplebelow describes another assay that was performed to evaluate the abilityof ribavirin to enhance or facilitate an immune response to an antigen.

EXAMPLE 13

This assay can be used with any ribavirin derivative or combinations ofribavirin derivatives to determine the extent that a particular vaccineformulation modulates a cellular immune response. To determine CD4⁺ Tcell responses to a ribavirin-containing vaccine, groups of mice wereimmunized s.c. with either 100 μg rNS3 in PBS or 100 μg rNS3 and 1 mgribavirin in PBS. The mice were sacrificed ten days post-immunizationand their lymph nodes were harvested and drained. In vitro recall assayswere then performed. (See e.g., Hultgren et al., J Gen Virol. 79:2381-91(1998) and Hultgren et al., Clin. Diagn. Lab. Immunol. 4:630-632 (1997),both of which are herein expressly incorporated by reference in theirentireties). The amount of CD4⁺ T cell proliferation was determined at96 h of culture by the incorporation of [³H] thymidine.

As shown in FIG. 5, mice that were immunized with 100 μg rNS3 mixed with1 mg ribavirin had a much greater T cell proliferative response thanmice that were immunized with 100 μg rNS3 in PBS. This data providesmore evidence that ribavirin enhances or facilitates a cellular immuneresponse (e.g., by promoting the effective priming of T cells).

Additional experiments were conducted to verify that ribavirin enhancesthe immune response to commercially available vaccine preparations. Theexample below describes the use of ribavirin in conjunction with acommercial HBV vaccine preparation.

EXAMPLE 14

The adjuvant effect of ribavirin was tested when mixed with two doses ofa commercially available vaccine containing HBsAg and alum. (Engerix,SKB). Approximately 0.2 μg or 2 μg of Engerix vaccine was mixed witheither PBS or 1 mg ribavirin in PBS and the mixtures were injected intraperitoneally into groups of mice (three per group). A booster containingthe same mixture was given on week four and all mice were bled on weeksix. The serum samples were diluted from 1:60 to 1:37500 and thedilutions were tested by EIA, as described above, except that purifiedhuman HBsAg was used as the solid phase antigen. As shown in TABLE 15,vaccine formulations having ribavirin enhanced the response to 2 μg ofan existing vaccine despite the fact that the vaccine already containedalum. That is, by adding ribavirin to a suboptimal vaccine dose (i.e.,one that does not induce detectable antibodies alone) antibodies becamedetectable, providing evidence that the addition of ribavirin allows forthe use of lower antigen amounts in a vaccine formulation withoutcompromising the immune response.

TABLE 15 End point antibody titer to HBsAg in EIA 0.02 μg Engerix 0.2 μgEngerix No ribavirin 1 mg ribavirin No ribavirin 1 mg ribavirin Week #1#2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 6 <60 <60 <60 <60 <60 <60 <60 <60 <60300 60 <60

The ribavirin used in the experiments above was obtained from commercialsuppliers (e.g., Sigma and ICN). The ribavirin that can be used with theembodiments described herein can also be obtained from commercialsuppliers or can be synthesized. The ribavirin and/or the antigen can beformulated with and without modification. For example, the ribavirin canbe modified or derivatized to make a more stable molecule and/or a morepotent adjuvant. By one approach, the stability of ribavirin can beenhanced by coupling the molecules to a support such as a hydrophilicpolymer (e.g., polyethylene glycol).

Many more ribavirin derivatives can be generated using conventionaltechniques in rational drug design and combinatorial chemistry. Forexample, Molecular Simulations Inc. (MSI), as well as many othersuppliers, provide software that allows one of skill to build acombinatorial library of organic molecules. The C2.Analog Builderprogram, for example, can be integrated with MSI's suite of Cerius2molecular diversity software to develop a library of ribavirinderivatives that can be used with the embodiments described herein.

By one approach, the chemical structure of ribavirin is recorded on acomputer readable media and is accessed by one or more modeling softwareapplication programs. The C2.Analog Builder program in conjunction withC2Diversity program allows the user to generate a very large virtuallibrary based on the diversity of R-groups for each substituentposition, for example. Compounds having the same structure as themodeled ribavirin derivatives created in the virtual library are thenmade using conventional chemistry or can be obtained from a commercialsource.

The newly manufactured ribavirin derivatives can then be screened inassays, which determine the extent of adjuvant activity of the moleculeand/or the extent of its ability to modulate of an immune response. Someassays may involve virtual drug screening software, such as C2.Ludi.C2.Ludi is a software program that allows a user to explore databases ofmolecules (e.g., ribavirin derivatives) for their ability to interactwith the active site of a protein of interest (e.g., RAC2 or another GTPbinding protein). Based upon predicted interactions discovered with thevirtual drug screening software, the ribavirin derivatives can beprioritized for further characterization in conventional assays thatdetermine adjuvant activity and/or the extent of a molecule to modulatean immune response. The section below provides more explanationconcerning the methods of using the compositions described herein.

Methods of Using the Vaccine Compositions

Routes of administration of the embodiments described herein include,but are not limited to, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Transdermal administration can beaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the adjuvant (e.g., ribavirin) and HCV antigen to penetrate theskin. Parenteral routes of administration include, but are not limitedto, electrical or direct injection such as direct injection into acentral venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Gastrointestinal routes ofadministration include, but are not limited to, ingestion and rectal.Transbronchial and transalveolar routes of administration include, butare not limited to, inhalation, either via the mouth or intranasally.

Compositions having the adjuvant (e.g., ribavirin) and HCV antigen thatare suitable for transdermal administration include, but are not limitedto, pharmaceutically acceptable suspensions, oils, creams, and ointmentsapplied directly to the skin or incorporated into a protective carriersuch as a transdermal device (“transdermal patch”). Examples of suitablecreams, ointments, etc. can be found, for instance, in the Physician'sDesk Reference. Examples of suitable transdermal devices are described,for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen,et al., herein expressly incorporated by reference in its entirety.

Compositions having the adjuvant (e.g., ribavirin) and HCV antigen thatare suitable for parenteral administration include, but are not limitedto, pharmaceutically acceptable sterile isotonic solutions. Suchsolutions include, but are not limited to, saline, phosphate bufferedsaline and oil preparations for injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection.

Compositions having the adjuvant (e.g., ribavirin) and HCV antigen thatare suitable for transbronchial and transalveolar administrationinclude, but not limited to, various types of aerosols for inhalation.Devices suitable for transbronchial and transalveolar administration ofthese are also embodiments. Such devices include, but are not limitedto, atomizers and vaporizers. Many forms of currently availableatomizers and vaporizers can be readily adapted to deliver vaccineshaving ribavirin and an antigen.

Compositions having the adjuvant (e.g., ribavirin) and HCV antigen thatare suitable for gastrointestinal administration include, but notlimited to, pharmaceutically acceptable powders, pills or liquids foringestion and suppositories for rectal administration.

The gene constructs described herein, in particular, may be administeredby means including, but not limited to, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gene guns”.Alternatively, the genetic vaccine may be introduced by various meansinto cells that are removed from the individual. Such means include, forexample, ex vivo transfection, electroporation, microinjection andmicroprojectile bombardment. After the gene construct is taken up by thecells, they are reimplanted into the individual. It is contemplated thatotherwise non-immunogenic cells that have gene constructs incorporatedtherein can be implanted into the individual even if the vaccinatedcells were originally taken from another individual.

According to some embodiments, the gene construct is administered to anindividual using a needleless injection device. According to someembodiments, the gene construct is simultaneously administered to anindividual intradermally, subcutaneously and intramuscularly using aneedleless injection device. Needleless injection devices are well knownand widely available. One having ordinary skill in the art can,following the teachings herein, use needleless injection devices todeliver genetic material to cells of an individual. Needleless injectiondevices are well suited to deliver genetic material to all tissue. Theyare particularly useful to deliver genetic material to skin and musclecells. In some embodiments, a needleless injection device may be used topropel a liquid that contains DNA molecules toward the surface of theindividual's skin. The liquid is propelled at a sufficient velocity suchthat upon impact with the skin the liquid penetrates the surface of theskin, permeates the skin and muscle tissue therebeneath. Thus, thegenetic material is simultaneously administered intradermally,subcutaneously and intramuscularly. In some embodiments, a needlelessinjection device may be used to deliver genetic material to tissue ofother organs in order to introduce a nucleic acid molecule to cells ofthat organ.

Preferred embodiments concern methods of treating or preventing HCVinfection. In these embodiments, an animal in need is provided an HCVantigen (e.g., a peptide antigen or nucleic acid-based antigen, asdescribed herein (SEQ. ID. NOs.: 1-27)) and an amount of adjuvant (e.g.,ribavirin) sufficient to exhibit an adjuvant activity in said animal.Accordingly, an animal can be identified as one in need by usingcurrently available diagnostic testing or clinical evaluation. Theadjuvant (e.g., ribavirin) and antigen can be provided separately or incombination, and other adjuvants (e.g., oil, alum, or other agents thatenhance an immune response) can also be provided to the animal in need.

Other embodiments of the invention include methods of enhancing animmune response to an HCV antigen by providing an animal in need with anamount of adjuvant (e.g., ribavirin) and one or more of SEQ. ID. NOs.:1-11, or a fragment thereof, preferably SEQ. ID. NOs.: 12-27 that iseffective to enhance said immune response. In these embodiments, ananimal in need of an enhanced immune response to an antigen isidentified by using currently available diagnostic testing or clinicalevaluation. By one approach, for example, an uninfected individual isprovided with the vaccine compositions described above in an amountsufficient to elicit a cellular and humoral immune response to NS3 so asto protect said individual from becoming infected with HCV. In anotherembodiment, an HCV-infected individual is identified and provided with avaccine composition comprising ribavirin and NS3 in an amount sufficientto enhance the cellular and humoral immune response against NS3 so as toreduce or eliminate the HCV infection. Such individual may be in thechronic or acute phase of the infection. In yet another embodiment, anHCV-infected individual suffering from HCC is provided with acomposition comprising an adjuvant (e.g., ribavirin) and the NS3/4Afusion gene in an amount sufficient to elicit a cellular and humoralimmune response against NS3-expressing tumor cells.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims. All referencescited herein are hereby expressly incorporated by reference.

1. A composition comprising a purified or isolated nucleic acid thatcomprises at least 100 consecutive nucleotides of SEQ. ID. No. 1 or thecomplement thereof, wherein said at least 100 consecutive nucleotideencodes a polypeptide comprising a NS3 domain of a fragment thereof. 2.The composition of claim 1, wherein said nucleic acid comprises at least200 consecutive nucleotides of SEQ. ID. No.
 1. 3. The composition ofclaim 1, wherein said nucleic acid comprises at least 500 consecutivenucleotides of SEQ. ID. No.
 1. 4. The composition of claim 1, whereinsaid nucleic acid comprises at least 1000 consecutive nucleotides ofSEQ. ID. No.
 1. 5. The composition of claim 1, wherein said nucleic acidcomprises a mutation that inhibits proteolytic cleavage of NS3 and NS4A.6. The composition of claim 2, wherein said nucleic acid comprises amutation that inhibits proteolytic cleavage of NS3 and NS4A.
 7. Thecomposition of claim 3, wherein said nucleic acid comprises a mutationthat inhibits proteolytic cleavage of NS3 and NS4A.
 8. The compositionof claim 4, wherein said nucleic acid comprises a mutation that inhibitsproteolytic cleavage of NS3 and NS4A.
 9. The composition of claim 1,wherein said nucleic acid encodes a polypeptide lacking a proteolyticcleavage site that inhibits proteolytic cleavage of NS3 and NS4A. 10.The composition of claim 1, comprising a vector that comprises saidnucleic acid.
 11. The composition of claim 2, comprising a vector thatcomprises said nucleic acid.
 12. The composition of claim 3, comprisinga vector that comprises said nucleic acid.
 13. The composition of claim4, comprising a vector that comprises said nucleic acid.
 14. Thecomposition of claim 5, comprising a vector that comprises said nucleicacid.
 15. The composition of claim 6, comprising a vector that comprisessaid nucleic acid.
 16. The composition of claim 7, comprising a vectorthat comprises said nucleic acid.
 17. The composition of claim 8,comprising a vector that comprises said nucleic acid.
 18. Thecomposition of claim 9, comprising a vector that comprises said nucleicacid.
 19. The composition of claim 1, further comprising an adjuvant.20. The composition of claim 2, further comprising an adjuvant.
 21. Thecomposition of claim 3, further comprising an adjuvant.
 22. Thecomposition of claim 4, further comprising an adjuvant.
 23. Thecomposition of claim 5, further comprising an adjuvant.
 24. Thecomposition of claim 6, further comprising an adjuvant.
 25. Thecomposition of claim 7, further comprising an adjuvant.
 26. Thecomposition of claim 8, further comprising an adjuvant.
 27. Thecomposition of claim 9, further comprising an adjuvant.
 28. Thecomposition of claim 1, further comprising ribavirin.
 29. Thecomposition of claim 2, further comprising ribavirin.
 30. Thecomposition of claim 3, further comprising ribavirin.
 31. Thecomposition of claim 4, further comprising ribavirin.
 32. Thecomposition of claim 5, further comprising ribavirin.
 33. Thecomposition of claim 6, further comprising ribavirin.
 34. Thecomposition of claim 7, further comprising ribavirin.
 35. Thecomposition of claim 8, further comprising ribavirin.
 36. Thecomposition of claim 9, further comprising ribavirin.
 37. Thecomposition of claim 1, wherein said composition is formulated for usewith an electroporation device.
 38. The composition of claim 1, whereinsaid composition is formulated for use with an injection device.
 39. Thecomposition of claim 38, wherein said injection device is a needlelessinjection device.
 40. The composition of claim 2, wherein saidcomposition is formulated for use with an electroporation device. 41.The composition of claim 2, wherein said composition is formulated foruse with an injection device.
 42. The composition of claim 41, whereinsaid injection device is a needleless injection device.
 43. Thecomposition of claim 3, wherein said composition is formulated for usewith an electroporation device.
 44. The composition of claim 3, whereinsaid composition is formulated for use with an injection device.
 45. Thecomposition of claim 44, wherein said injection device is a needlelessinjection device.
 46. The composition of claim 4, wherein saidcomposition is formulated for use with an electroporation device. 47.The composition of claim 4, wherein said composition is formulated foruse with an injection device.
 48. The composition of claim 47, whereinsaid injection device is a needleless injection device.
 49. Thecomposition of claim 5, wherein said composition is formulated for usewith an electroporation device.
 50. The composition of claim 5, whereinsaid composition is formulated for use with an injection device.
 51. Thecomposition of claim 50, wherein said injection device is a needlelessinjection device.
 52. The composition of claim 6, wherein saidcomposition is formulated for use with an electroporation device. 53.The composition of claim 6, wherein said composition is formulated foruse with an injection device.
 54. The composition of claim 53, whereinsaid injection device is a needleless injection device.
 55. Thecomposition of claim 7, wherein said composition is formulated for usewith an electroporation device.
 56. The composition of claim 7, whereinsaid composition is formulated for use with an injection device.
 57. Thecomposition of claim 56, wherein said injection device is a needlelessinjection device.
 58. The composition of claim 8, wherein saidcomposition is formulated for use with an electroporation device. 59.The composition of claim 8, wherein said composition is formulated foruse with an injection device.
 60. The composition of claim 59, whereinsaid injection device is a needleless injection device.
 61. Thecomposition of claim 9, wherein said composition is formulated for usewith an electroporation device.
 62. The composition of claim 9, whereinsaid composition is formulated for use with an injection device.
 63. Thecomposition of claim 62, wherein said injection device is a needlelessinjection device.
 64. A composition comprising a purified or isolatednucleic acid that comprises at least 100 consecutive nucleotidesencoding a peptide sequence of SEQ. ID. NO:
 2. 65. The composition ofclaim 1, wherein said nucleic acid comprises at least 500 consecutivenucleotides encoding the peptide sequence of SEQ. ID. NO:
 2. 66. Thecomposition of claim 64, further comprising an adjuvant.
 67. Thecomposition of claim 64, further comprising ribavirin.
 68. Thecomposition of claim 64, wherein said composition is formulated for usewith an electroporation device.
 69. The composition of claim 64, whereinsaid composition is formulated for use with an injection device.
 70. Thecomposition of claim 69, wherein said injection device is a needlelessinjection device.